Methods and compositions involving promoters derived from Yarrowia lipolytica

ABSTRACT

The current methods and compositions provide for nucleotide sequences of promoters from  Yarrowia lipolytica  which may be used to drive gene expression in a cell. In some aspects, the promoters are useful for modulating lipid production in oleaginous organisms such as yeast.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Application No.62/879,989, filed Jul. 29, 2019, incorporated by reference herein in itsentirety.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted in ASCII format and is hereby incorporated by reference in itsentirety. Said ASCII copy, created on Jul. 29, 2020, is namedNOVG_P0016US_Seq.txt and is 64,078 bytes in size.

BACKGROUND Field of the Invention

The disclosure is generally directed to biotechnology. Embodimentspertain to promoter regions from Yarrowia lipolytica that direct geneexpression of other genes and/or in other organisms.

Description of Related Art

Oleaginous yeasts, such as Yarrowia lipolytica, may be engineered forthe industrial production of lipids, which are indispensable ingredientsin the food and cosmetics industries, and important precursors in thebiodiesel and biochemical industries. The lipid yield and composition(i.e. the types of fatty acids in the lipids) of an oleaginous organismcan be increased by up-regulating or down-regulating the genes thatregulate cellular metabolism and lipid pathways.

Some Y. lipolytica promoters have been identified and validated (See,e.g., U.S. Pat. Nos. 7,259,255; 7,264,949; U.S. Patent Publication No.2012/0289600; US Patent Publication No. 2006/0094102; and Wartmann etal., FEMS YEAST RESEARCH 2:363-69 (2002), all of which are herewithincorporated by reference in their entirety). Yarrowia, however,contains hundreds of promoters that have yet to be identified, and manyof these promoters may be useful for engineering yeast and otherorganisms. A promoter may vary considerably between different strains ofthe same species, and the identification and screening of such geneticpolymorphisms provides a richer toolbox for genetic engineering.

One approach to up-regulating a gene is to control its expression usinga constitutive promoter. For example, the Y. lipolytica diacylglycerolacyltransferase DGA1 may be up-regulated using a strong constitutivepromoter (See, e.g., Tai & Stephanopoulos, METABOLIC ENGINEERING 15:1-9(2013)).

Choosing optimal promoters for controlling gene expression is a criticalpart of genetic engineering, but different promoters may be optimal fordifferent applications. For example, the optimal promoters for anindustrial strain of yeast may not be the same as promoters that areoptimal in laboratory strains.

There remains a need for efficient yeast systems that control variousphenotypes, for example, increase or reduction in lipid production andmodification of lipid composition.

SUMMARY OF THE DISCLOSURE

Here, embodiments include, inter alia, promoter sequences comprising SEQID NO:1, 2, 5, 6, 7, or subsequences thereof, that are useful fordifferential expression of genes of a host cell. The promoters maypromote decreased expression of a coding sequence during a lipidaccumulation phase of Yarrowia lipolytica relative to a growth phase. Insome embodiments, the use of such promoters is advantageous when thereis a need to eliminate or reduce protein accumulation and/or activityduring the lipid production phase of an oleaginous host cell, whilemaintaining some level of gene expression and/or protein activity duringthe growth of the cell. This differential expression of genes during thegrowth cycle of an organism can be used, for example, to optimize lipidyield, lipid composition, or the efficiency of the industrial processinvolving the organism. A gene, or a sequence of interest such as acoding sequence, can be placed under the control of a promoter sequencecomprising SEQ ID NO:1, 2, 5, 6, or 7 or a subsequence thereof to allowtranscription of the coding sequence and activity of the resultantprotein during an active growth phase of a host cell, while reducingtranscription of the coding sequence and/or activity of the resultprotein during a lipid accumulation phase.

Embodiments include compositions comprising one or more nucleic acidmolecules. Embodiments include a nucleic acid molecule comprising one ormore sequences (e.g., promoter sequences, coding sequences, etc.).Embodiments also include recombinant, transformed or modified cells,vectors, and/or expression cassettes comprising such nucleic acidmolecules.

Embodiments also include methods of expressing a sequence of interest(e.g., a coding sequence), methods of transcribing a sequence, method ofregulating transcription, methods of regulating expression, methods ofmodulating lipid production in a host cell, methods of making a nucleicacid comprising a promoter sequence, methods of using a nucleic acidcomprising a promoter sequence, methods of linking two sequences (e.g.,a promoter sequence and a coding sequence), methods of regulatingexpression of a sequence, methods of regulating the activity of asequence, methods of expressing a sequence in a cell, methods ofreducing expression of a sequence in a lipid accumulation phase relativeto a growth phase, methods of optimizing lipid production, methods ofincreasing lipid yield, methods of modifying lipid composition of acell, methods of culturing a host cell, methods of fermentation, methodsof producing lipids, methods of differentially expressing a polypeptide,and improvements of known methods for producing lipids. The steps andembodiments discussed in this disclosure are contemplated as part of anyof these methods. The methods, steps and embodiments discussed in thisdisclosure regarding Yarrowia lipolytica are also contemplated for othermicrobial cells, yeast cells, fungal cells and plant cells. In someembodiments, the methods contemplated here can comprise or exclude anyof the following steps: providing a nucleic acid, operably linking to acoding sequence a heterologous nucleic acid sequence, introducing arecombinant coding sequence into a host cell, expressing a codingsequence, generating a nucleic acid molecule comprising a sequencecomprising a promoter sequence operably linked to a coding sequence,subjecting a cell to conditions sufficient to express a sequence (e.g.,a coding sequence), introducing a promoter sequence into a nucleic acidmolecule, and/or culturing a host cell.

In some aspects, the disclosure relates to a nucleic acid moleculecomprising a first sequence having at least 70% sequence identity to SEQID NO:1, 2, 5, 6, or 7. In some embodiments, the first sequence has atleast 80% sequence identity to SEQ ID NO:1, 2, 5, 6, or 7. In someembodiments, the first sequence has 70%, 71%, 72%, 73%, 74%, 75%, 76%,77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity, or anyrange derivable therein, to SEQ ID NO: 1, 2, 5, 6, or 7. In someembodiments, the first sequence is SEQ ID NO:1, 2, 5, 6 or 7. In someembodiments, the first sequence is linked to a second sequence. In someembodiments, the first sequence is operably linked to a second sequence.In some embodiments, the second sequence is heterologous to the firstsequence. In some embodiments the second sequence is from Yarrowia. Insome embodiments, the second sequence is from Yarrowia lipolytica. Yet,in some embodiments, the second sequence is not from Yarrowia.

In some aspects, the disclosure relates to a nucleic acid moleculecomprising a first sequence having at least 70% sequence identity to asubsequence of SEQ ID NO: 1, 2, 5, 6 or 7. In some embodiments, thefirst sequence has at least 80% sequence identity to a subsequence ofSEQ ID NO:1, 2, 5, 6, or 7. In some embodiments, the first sequence has70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%,84%, 85%, 86%, 87%, 88%, 89%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99% or 100% sequence identity, or any range derivable therein, to asubsequence of SEQ ID NO: 1, 2, 5, 6, or 7. In some embodiments, thefirst sequence is operably linked to a second sequence. In someembodiments, the second sequence is heterologous to the first sequence.In some embodiments, the first sequence is capable of conferringexpression of the second sequence. In some embodiments, the secondsequence is a coding sequence. In some embodiments, the first sequenceis constructed from various promoter functional elements derived fromany of SEQ ID NO:1, 2, 5, 6 or 7.

In some aspects, the disclosure relates to vectors comprising anucleotide sequence disclosed herein, for example, a sequence having atleast 70% sequence identity to SEQ ID NO:1, 2, 5, 6 or 7 or asubsequence of SEQ ID NO:1, 2, 5, 6 or 7 or a functional variant of SEQID NO:1, 2, 5, 6 or 7. In some embodiments, the vector further comprisesa coding sequence. In some embodiments the vector is a plasmid. In otherembodiments, the vector is a linear DNA molecule.

Further aspects of the disclosure relate to a cell comprising a nucleicacid molecule comprising SEQ ID NO: 1, 2, 3, 4, 5, 6 or 7 or a sequencethat is 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%,83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99% or 100% (or any range derivable herein) identical to SEQ IDNO:1, 2, 5, 6 or 7 or a subsequence of SEQ ID NO:1, 2, 5, 6 or 7 or afunctional variant of SEQ ID NO:1, 2, 5, 6 or 7. In some embodiments,the cell further comprises a coding sequence. In some embodiments, thenucleic acid molecule is a genome of the cell. In some embodiments, thenucleic acid molecule is a vector within the cell. In some embodiments,the cell is a yeast cell. In some embodiments, the yeast cell is anArxula, Aspergillus, Aurantiochytrium, Candida, Claviceps, Cryptococcus,Cunninghamella, Geotrichum, Hansenula, Kluyveromyces, Kodamaea,Leucosporidiella, Lipomyces, Mortierella, Ogataea, Pichia, Prototheca,Rhizopus, Rhodosporidium, Rhodotorula, Saccharomyces,Schizosaccharomyces, Tremella, Trichosporon, Wickerhamomyces, orYarrowia cell. In some embodiments, the cell is an Aspergillus niger,Aspergillus orzyae, Aspergillus terreus, Aurantiochytrium limacinum,Candida utilis, Claviceps purpurea, Cryptococcus albidus, Cryptococcuscurvatus, Cryptococcus ramirezgomezianus, Cryptococcus terreus,Cryptococcus wieringae, Cunninghamella echinulata, Cunninghamellajaponica, Geotrichum fermentans, Hansenula polymorpha, Kluyveromyceslactis, Kluyveromyces marxianus, Kodamaea ohmeri, Leucosporidiellacreatinivora, Lipomyces lipofer, Lipomyces starkeyi, Lipomycestetrasporus, Mortierella isabellina, Mortierella alpina, Ogataeapolymorpha, Pichia ciferrii, Pichia guilliermondii, Pichia pastoris,Pichia stipites, Prototheca zopfii, Rhizopus arrhizus, Rhodosporidiumbabjevae, Rhodosporidium toruloides, Rhodosporidium paludigenum,Rhodotorula glutinis, Rhodotorula mucilaginosa, Saccharomycescerevisiae, Schizosaccharomyces pombe, Tremella enchepala, Trichosporoncutaneum, Trichosporon fermentans, Wickerhamomyces ciferrii, or aYarrowia lipolytica cell.

Additional aspects of the disclosure relate to a method of expressing acoding sequence in a cell comprising culturing a cell comprising anucleic acid molecule comprising the coding sequence, wherein the codingsequence is operably linked to a promoter comprising i) at least 80%sequence identity to SEQ ID NO: 1, 2, 5, 6, or 7; ii) a variant of SEQID NO: 1, 2, 5, 6, or 7, or iii) a subsequence of SEQ ID NO: 1, 2, 5, 6,or 7. In some embodiments, the method further comprises subjecting thecell to conditions sufficient to express the coding sequence. In someembodiments, conditions sufficient to express the coding sequenceinclude, for example, culturing the cell, providing nutrients to thecell, and providing one or more growth factors to the cell.

Further aspects of the disclosure relate to a method of linking apromoter sequence to a coding sequence comprising (a) providing (I) afirst nucleic acid molecule comprising a promoter sequence comprising i)at least 80% sequence identity to SEQ ID NO: 1, 2, 5, 6, or 7; ii) avariant of SEQ ID NO: 1, 2, 5, 6, or 7, or iii) a subsequence of SEQ IDNO: 1, 2, 5, 6, or 7; and (II) a second nucleic acid molecule comprisinga coding sequence; and (b) using the first nucleic acid molecule and thesecond nucleic acid molecule to generate a third nucleic acid moleculecomprising the promoter sequence operably linked to the coding sequence.In some embodiments of the method, the promoter is 70%, 71%, 72%, 73%,74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%,88%, 89%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% (or anyrange derivable herein) identical to SEQ ID NO:1, 2, 5, 6 or 7, or asubsequence thereof.

Further aspects of the disclosure involve methods of increasing thelipid yield of a recombinant cell during a fermentation process, whereinthe fermentation process comprises a growth phase and a lipidaccumulation phase. In some embodiments, the method comprises:introducing into a cell a nucleic acid molecule comprising a promotersequence operably linked to a coding sequence, wherein the promotersequence comprises i) a nucleotide sequence having at least 80% sequenceidentity to SEQ ID NO: 1, 2, 5, 6, or 7, ii) a functional variant of SEQID NO: 1, 2, 5, 6, or 7, or iii) a subsequence of SEQ ID NO: 1, 2, 5, 6,or 7, thereby obtaining a recombinant cell and culturing the recombinantcell; and culturing the recombinant cell, wherein the lipid yield of therecombinant cell is increased as compared to a non-recombinant cell ofthe same species. In some embodiments, the method comprises (a)introducing into a cell a first nucleic acid molecule comprising apromoter sequence comprising i) a sequence having at least 80% sequenceidentity to SEQ ID NO: 1, 2, 5, 6, or 7, ii) a sequence comprising afunctional variant of SEQ ID NO: 1, 2, 5, 6, or 7, or iii) a sequencehaving at least 80% identity to a subsequence of SEQ ID NO: 1, 2, 5, 6,or 7, thereby obtaining a recombinant cell; (b) in the cell, using thefirst nucleic acid molecule and a second nucleic acid moleculecomprising a coding sequence to generate a third nucleic acid moleculecomprising the promoter sequence operably linked to the coding sequence;and (c) culturing the recombinant cell, wherein the lipid yield of therecombinant cell is increased as compared to a non-recombinant cell ofthe same species. In some embodiments, the promoter confers increasedexpression or activity of the coding sequence during the growth phase ascompared to the lipid accumulation phase.

Additional aspects of the disclosure relate to a method of regulatingthe expression or activity of a coding sequence during a fermentationprocess wherein the fermentation process comprises a growth phase and alipid accumulation phase. In some embodiments, the method comprises: (a)introducing a nucleic acid molecule into a cell, wherein the nucleicacid molecule comprises the coding sequence operably linked to apromoter sequence, wherein the promoter sequence comprises i) a sequencehaving at least 80% sequence identity to SEQ ID NO: 1, 2, 5, 6, or 7,ii) a sequence comprising a functional variant of SEQ ID NO: 1, 2, 5, 6,or 7 or iii) a sequence having at least 80% sequence identity to asubsequence of SEQ ID NO: 1, 2, 5, 6, or 7; and (b) culturing the cell,thereby regulating expression or activity of the coding sequence. Insome embodiments, the method comprises (a) providing a first nucleicacid molecule to a cell, wherein the first nucleic acid comprises apromoter sequence comprising i) a sequence having at least 80% sequenceidentity to SEQ ID NO: 1, 2, 5, 6, or 7, ii) a sequence comprising afunctional variant of SEQ ID NO: 1, 2, 5, 6, or 7 or iii) a sequencehaving at least 80% identity to a subsequence of SEQ ID NO: 1, 2, 5, 6,or 7; (b) in the cell, using the first nucleic acid molecule and asecond nucleic acid molecule comprising a coding sequence to generate athird nucleic acid molecule comprising the promoter sequence operablylinked to the coding sequence; and (c) culturing the cell, therebyregulating expression or activity of the coding sequence.

In some embodiments the promoter sequence confers decreased expressionor activity of the coding sequence during the lipid accumulation phaseas compared to the growth accumulation phase. In some embodiments, theexpression or activity of the coding sequence is reduced by at least50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%,64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%,78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more in the lipidaccumulation phase as compared to the growth phase. In some embodiments,the cell is subjected to fermentation conditions, where the expressionor activity of the coding sequence is reduced 96 hours after subjectingthe cell to the fermentation conditions relative to 16 hours aftersubjecting the cell to the fermentation conditions. In some embodiments,the fermentation conditions comprise microaerobic conditions. In someembodiments, the expression or activity of the coding sequence isreduced 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39,40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57,58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75,76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93,94, 95, or 96 hours after providing the first nucleic acid molecule tothe cell relative to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, or 22 hours after providing the first nucleicacid molecule to the cell. In some embodiments, the method furthercomprises operably linking the coding sequence to the promoter sequence.

The term “growth phase” refers to a phase during which cells aredividing and creating catalytic biomass in non-limited nutrientconditions. The term “lipid accumulation phase” refers to a phase duringwhich there is a decrease in growth rate due to nutrient limitation andexcess carbon is diverted to lipid production.

Further aspects relate to a method of making a nucleic acid capable ofexpressing a coding sequence in a cell. In some embodiments, the methodcomprises: (a) introducing into the cell a nucleic acid moleculecomprising a coding sequence, wherein the coding sequence is operablylinked to a promoter sequence comprising i) at least 80% sequenceidentity to SEQ ID NO: 1, 2, 5, 6, or 7; ii) a variant of SEQ ID NO: SEQID NO: 1, 2, 5, 6, or 7, or iii) a subsequence of SEQ ID NO: 1, 2, 5, 6,or 7; and (b) subjecting the cell to conditions sufficient to expressthe coding sequence. In some embodiments, the method comprises (a)introducing into the cell a nucleic acid molecule comprising a promotersequence comprising i) a sequence having at least 80% sequence identityto SEQ ID NO: 1, 2, 5, 6, or 7; ii) a sequence comprising a functionalvariant of SEQ ID NO: SEQ ID NO: 1, 2, 5, 6, or 7, or iii) a sequencecomprising at least 80% identity to a subsequence of SEQ ID NO: 1, 2, 5,6, or 7; (b) in the cell, using the first nucleic acid molecule and asecond nucleic acid molecule comprising a coding sequence to generate athird nucleic acid molecule comprising the promoter sequence operablylinked to the coding sequence; and (c) subjecting the cell to conditionssufficient to express the coding sequence. In some embodiments, themethod further comprises operably linking the coding sequence to theheterologous nucleic acid sequence.

In some embodiments of the disclosed methods, using a first nucleic acidmolecule and a second nucleic acid molecule to generate a third nucleicacid molecule comprising a promoter sequence operably linked to a codingsequence comprises introducing the promoter sequence into the secondnucleic acid molecule. In some embodiments, the promoter sequence isintroduced into the second nucleic acid molecule upstream of the codingsequence. In some embodiments, the promoter sequence is introduced intothe second nucleic acid molecule by recombination. In some embodiments,the coding sequence is heterologous to the promoter sequence. In someembodiments, the first nucleic acid molecule is a vector. In someembodiments, the second nucleic acid molecule is a vector. In someembodiments, the second nucleic acid molecule is a nucleic acid moleculeof a genome of a cell. In some embodiments, the method further comprisesintroducing the third nucleic acid molecule to a cell. In someembodiments, the method further comprises subjecting the cell toconditions sufficient to express the coding sequence.

It is contemplated that any method or composition described herein canbe implemented with respect to any other method or composition describedherein and that different embodiments may be combined.

Use of the one or more sequences or compositions may be employed basedon any of the methods described herein. Other embodiments are discussedthroughout this application. Any embodiment discussed with respect toone aspect of the disclosure applies to other aspects of the disclosureas well and vice versa. For example, any step in a method describedherein can apply to any other method. Moreover, any method describedherein may have an exclusion of any step or combination of steps. Theembodiments in the Example section are understood to be embodiments thatare applicable to all aspects of the technology described herein.

Other objects, features and advantages of the present invention willbecome apparent from the following detailed description. It should beunderstood, however, that the detailed description and the specificexamples, while indicating preferred embodiments of the invention, aregiven by way of illustration only, since various changes andmodifications within the spirit and scope of the invention will becomeapparent to those skilled in the art from this detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings form part of the present specification and areincluded to further demonstrate certain aspects of the presentinvention. The invention may be better understood by reference to one ormore of these drawings in combination with the detailed description ofspecific embodiments presented herein.

FIG. 1 depicts the DNA sequence of SEQ ID NO:1, also referred to aspromoter PR104 (YALI0E24189g, from −1000 to −1).

FIG. 2 depicts the DNA sequence of SEQ ID NO:2, also referred to aspromoter PR105 (YALI0B16522g, from −1000 to −1).

FIG. 3 depicts the DNA sequence of SEQ ID NO:3, also referred to aspromoter PR106 (YALI0B21010g, from −1000 to −1).

FIG. 4 depicts the DNA sequence of SEQ ID NO:4 also referred to aspromoter PR107 (YALI0C01411g, from −1000 to −1).

FIG. 5 depicts the DNA sequence of SEQ ID NO:5 also referred to aspromoter PR108 (YALI0A04631g, from −370 to −1).

FIG. 6 depicts the DNA sequence of SEQ ID NO:6 also referred to aspromoter PR109 (YALI0A12815g, from −1000 to-1).

FIG. 7 depicts the DNA sequence of SEQ ID NO:7 also referred to aspromoter PR110 (YALI0B19800g, from −1000 to-1).

FIGS. 8A-8C show results from experiments described in Example 1. FIG.8A shows lipid compositions at the indicated time points for Y.lipolytica cells expressing FAD2 controlled by one of 7 promoters(PR104-PR107) compared with a wild-type strain (YB-392) and a Δfad2strain (NS419). FIG. 8B shows linoleate production for the same timepoints and cells as FIG. 8A. FIG. 8C shows example microscopicobservations of each cell type at 96 hours.

FIGS. 9A-9C show results from experiments described in Example 1. FIG.9A shows lipid compositions at the indicated time points for Y.lipolytica cells expressing OLE1 controlled by one of 4 promoters(PR104, PR105, PR109, and PR110) compared with a wild-type strain(YB-392). FIG. 9B shows OLE1 product production for the same time pointsand cells as FIG. 9A. FIG. 9C shows example microscopic observations ofeach cell type at 96 hours.

SEQUENCE LISTING

The instant application contains a Sequence Listing that has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Jul. 29, 2020 isnamed NOVG_P0016US_Seq.txt and is 9,318 bytes in size.

DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

Increasing efficiency and maximizing lipid metabolism is an importantfactor in large scale, industrial production of lipids. Researchers haveattempted various methods to eliminate or reduce high protein activityduring the lipid accumulation phase while maintaining protein activityduring growth. One method included gene deletion. Gene deletion,however, has a drawback. It deprives the cell of the correspondinggene/protein activity during the growth phase. Another method is tooverexpress a desired activity during lipid accumulation so as tooutcompete the unwanted activity. This method is partially effective atbest due to the persistence of the undesired protein(s). There existpromoters that can be turned on or off using a culture additive or otherexternal trigger such as galactose induction or erythritol induction(Trassaert et al 2017 New inducible promoter for gene expression andsynthetic biology in Yarrowia lipolytica. Microb Cell Fact. 16(1):141).However, this latter method requires a process change, often limits theculture media that can be used and adds to the overall cost of thefermentation process.

Accumulation of lipid in Y. lipolytica may be triggered by nutrientlimitation in the presence of excess carbon. Nitrogen limitation iscommonly used for lipid accumulation studies because it is an easilycontrolled parameter. A Y. lipolytica lipid fermentation may comprisetwo phases: a) growth phase where there is enough nitrogen present forthe cells to divide and produce catalytic biomass and, b) lipidproduction phase where nutrient limitation occurs causing a decrease ingrowth rate and activation of lipid production. Some pathways (e.g.,nucleic acid and protein synthesis) are repressed and others areactivated (e.g., fatty acid and triacylglycerol (TAG) synthesis). Y.lipolytica cultivations for lipid production may last at least 4 daysand involve a high carbon to nitrogen ratio (C/N) such as 50:1 or 75:1.

Through analysis of publicly available transcriptomic data, Yarrowialipolytica transcripts were identified whose abundance dropped at highcarbon-to-nitrogen (C:N) ratios. Transcript levels were compared betweenchemostat growth and D-stat cultivation when C:N ratio reached 25. Thepromoter regions of these genes were designated as PR104-PR110 (SEQ IDNOs:1-7). The inventors took the 1000 bp (or 370 bp for PR108 (SEQ IDNO:5) where the intergenic region is shorter) upstream of thetranscription start site of each of the corresponding genes and usedthem to drive transcription of genes of interest in order to obtainactivity during growth but not during lipid accumulation (a transitioninvolving an increase in the carbon-to-nitrogen ratio). Descriptions ofthe promoters designated PR104-PR110 are provided in Table 1.

TABLE 1 Description of promoters Ratio (transcript in chemostat/Associated Annotation using Pfam Motif transcript at Promoter gene SizeBLAST/KEGG (KEGG) high C:N) PR104 YALI0E24189g −1000 to −1 hypotheticalprotein F-box-like; F- 100 conserved in the box_4; F-box Yarrowia clade(no hits by homology) PR105 YALI0B16522g −1000 to −1 yeast amino acidAA_permease;  51 transporter (similar to AA_permease_2 uniprot|P19145Saccharomyces cerevisiae YKR039w GAP1 general amino acid permease) PR106YALI0B21010g −1000 to −1 similar to Candida MFS_1;  40 albicans putativeHerpes_gE; allantoate permease V_ATPase_I; (by homology) DUF4500 PR107YALI0C01411g −1000 to −1 1,3-beta-glucan Glucan_synthase;  34 synthaseFKS1_dom1 [EC:2.4.1.34]; similar to uniprot|Q6C549 Yarrowia lipolyticaYALI0E21021g FKS1 component of 1,3-beta- glucan synthase PR108YALI0A04631g  −370 to −1 weakly similar to F-box-like  32 uniprot|Q6C9E1Yarrowia lipolytica YALI0D11924g, protein with a F-box motif Product:Hypothetical protein of a 30-member gene family, conserved in theYarrowia clade. PR109 YALI0A12815g −1000 to −1 hypothetical protein  28conserved in the Yarrowia clade PR110 YALI0B19800g −1000 to −1 similarto AA_permease;  20 uniprot|P19145 AA_permease_2 Saccharomycescerevisiae YKR039W GAP1 General amino acid permease

Embodiments herein include placing a sequence or a gene of interestunder the control of one of a set of promoters comprising SEQ ID NO:1,2, 5, 6 or 7 or variants or subsequences thereof that facilitate reducedexpression or activity of the sequence or gene of interest during alipid accumulation phase relative to a growth phase. If the generepresents a native gene, the native locus may be deleted. In somecases, a promoter may be introduced into nucleic acid of a cell, therebydriving expression of a native locus. Differential expression might beused to optimize lipid yield or composition. A key feature ofembodiments of the current disclosure is the ability to modulateexpression of genes without changing the fermentation process (noadditives are required) and that it is tied into the established processof lipid production of Yarrowia lipolytica.

The meaning of terms as intended is defined herein below.

I. Definitions

The term “expression” refers to the amount of a nucleic acid or aminoacid sequence (e.g., peptide, polypeptide, or protein) in a cell. Theincreased expression of a gene refers to the increased transcription ofthat gene. The increased expression of an amino acid sequence, peptide,polypeptide, or protein refers to the increased translation of a nucleicacid encoding the amino sequence, peptide, polypeptide, or protein.Expression may refer to secreted or non-secreted expression products,including polypeptides or metabolites.

The term “expression system” or “expression cassette” refers to nucleicacid molecules containing a desired coding sequence and controlsequences in operable linkage, so that hosts transformed or transfectedwith these sequences are capable of producing the encoded proteins orhost cell metabolites. In order to effect transformation, the expressionsystem may be included in a vector; however, the relevant DNA may alsobe integrated into the host chromosome.

“Expression constructs” or “vectors” or “plasmid” refer to DNA sequencesthat are required for the transcription of cloned recombinant nucleotidesequences, i.e. of recombinant genes and the translation of their mRNAin a suitable host organism. Expression vectors or plasmids usuallycomprise an origin for autonomous replication in the host cells,selectable markers (e.g. an amino acid synthesis gene or a geneconferring resistance to antibiotics such as zeocin, kanamycin, G418 orhygromycin), a number of restriction enzyme cleavage sites, a suitablepromoter sequence and a transcription terminator, which components areoperably linked together. The terms “plasmid” and “vector” as usedherein include autonomously replicating nucleotide sequences as well asgenome integrating nucleotide sequences. The expression construct of thedisclosure specifically comprises a promoter of the disclosure, operablylinked to a nucleotide sequence encoding a polypeptide under thetranscriptional control of the promoter, which promoter is not nativelyassociated with the coding sequence.

The term “coding sequence” refers to a DNA sequence which codes for aspecific amino acid sequence. The terms “coding sequence” and “codingregion” are used interchangeably herein. A “coding region of interest”is a coding region which is desired to be expressed.

“Regulatory sequences” refer to nucleotide sequences located upstream(5′ non-coding sequences), within, or downstream (3′ non-codingsequences) of a coding sequence, and which influence the transcription,RNA processing or stability, or translation of the associated codingsequence. Regulatory sequences may include, but are not limited to:promoters, enhancers, silencers, 5′ untranslated leader sequence (e.g.,between the transcription start site and translation initiation codon),introns, polyadenylation recognition sequences, RNA processing sites,effector binding sites and stem-loop structures.

The term “heterologous” as used herein with respect to a nucleotide oramino acid sequence refers to a sequence that is not found in aparticular context in nature. An example of a heterologous sequence is anucleotide sequence not natively associated with the promoter accordingto the disclosure; in some embodiments, a promoter directs or controlsthe expression of a sequence (i.e., produces an RNA transcript) whoseexpression is not directed or controlled by that promoter in nature. Apolynucleotide comprising a promoter region connected to a heterologoussequence would be considered a hybrid or chimeric polynucleotide. Apromoter may be linked to a heterologous sequence from the same organism(e.g., a sequence that is not the same sequence whose expression iscontrolled by the promoter, such as from a different region of thegenome) or a promoter may be linked to a heterologous sequence from adifferent organism. In some embodiments, the promoter controls ordirects the expression of a different gene or coding sequence than itcontrols or directs in nature.

The term “variant”, “functional variant” or “functional promotervariant” as used herein refers to any sequence with a specific sequenceidentity to a comparable parent sequence. The term can include anysequence derived from SEQ ID NOs:1-7, including sequences that arerecombinantly engineered from various promoter elements or promoterregions derived from SEQ ID NOs:1-7 to functionally effect thetranscription of an operably linked coding sequence. A variant,functional variant or functional promoter variant may be derived from aparent sequence e.g., by size variation, such as (terminal ornon-terminal, such as “interstitional” i.e. with deletions or insertionswithin the nucleotide sequence) elongation, fragmentation, imitation,hybridization (including combination of sequences). A “functionalvariant” or a “functional promoter variant” is any variant that retainsthe ability to drive expression of an operably attached coding sequence.

The term “gene,” as used herein, may encompass genomic sequences thatcontain introns, particularly polynucleotide sequences encodingpolypeptide sequences involved in a specific activity. The term furtherencompasses synthetic nucleic acids that did not derive from genomicsequence. In certain embodiments, the genes lack introns, as they aresynthesized based on the known DNA sequence of cDNA and protein sequenceor because the gene does not have any introns in nature. In otherembodiments, the genes are synthesized, non-native cDNA wherein thecodons have been optimized for expression in Y. lipolytica based oncodon usage. The term can further include nucleic acid moleculescomprising upstream, downstream, and/or intron nucleotide sequences,including promoters.

The term “genetic modification” refers to the result of atransformation. Every transformation causes a genetic modification bydefinition.

The term “homolog,” as used herein, refers to (a) nucleic acids havingnucleotide substitutions, deletions and/or insertions relative to theunmodified nucleic acid in question and having similar biological andfunctional activity as the unmodified nucleic acid from which they arederived, and (b) peptides, oligopeptides, polypeptides, proteins, andenzymes having amino acid substitutions, deletions and/or insertionsrelative to the unmodified protein in question and having similarbiological and functional activity as the unmodified protein from whichthey are derived.

The term “integrated” refers to a nucleic acid that is maintained in acell as an insertion into the genome of the cell, such as insertion intoa chromosome, including insertions into a plastid genome.

The term “introducing” such as in “introducing a coding sequence into acell” refers to any means by which a heterologous or native nucleic acidis brought into a cell so as to genetically modify or engineer the cell.

The term “vector” refers to the means by which a nucleic acid can bepropagated and/or transferred between organisms, cells, or cellularcomponents. Vectors include plasmids, linear DNA fragments, viruses,bacteriophage, pro-viruses, phagemids, transposons, and artificialchromosomes, and the like, that may or may not be able to replicateautonomously or integrate into a chromosome of a host cell.

“In operable linkage” “operably linked” is a functional linkage betweentwo nucleic acid sequences, such a control sequence (typically apromoter) and the linked sequence (typically, a sequence that encodes aprotein, also called a coding sequence). A promoter is in operablelinkage (or “operably linked”) with a gene if it can mediatetranscription of the gene.

The term “native” refers to the composition of a cell or parent cellprior to a transformation event.

The terms “nucleic acid” refers to a polymeric form of nucleotides ofany length, either deoxyribonucleotides or ribonucleotides, or analogsthereof. Polynucleotides may have any three-dimensional structure, andmay perform any function. The following are non-limiting examples ofpolynucleotides: coding or non-coding regions of a gene or genefragment, loci (locus) defined from linkage analysis, exons, introns,messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozymes, cDNA,recombinant polynucleotides, branched polynucleotides, plasmids,vectors, isolated DNA of any sequence, isolated RNA of any sequence,nucleic acid probes, and primers. A polynucleotide may comprise modifiednucleotides, such as methylated nucleotides and nucleotide analogs. Ifpresent, modifications to the nucleotide structure may be impartedbefore or after assembly of the polymer. A polynucleotide may be furthermodified, such as by conjugation with a labeling component. In allnucleic acid sequences provided herein, U nucleotides areinterchangeable with T nucleotides.

The term “parent cell” refers to every cell from which a cell descended.The genome of a cell is comprised of the parent cell's genome and anysubsequent genetic modifications to its genome.

As used herein, the term “plasmid” refers to a circular DNA moleculethat is physically separate from an organism's genomic DNA. Plasmids maybe linearized before being introduced into a host cell (referred toherein as a linearized plasmid). Linearized plasmids may not beself-replicating, but may integrate into and be replicated with thegenomic DNA of an organism.

A “promoter” is a nucleic acid region that directs or controlstranscription of a nucleic acid sequence on the same polynucleotide. Insome embodiments, a promoter directs transcription of an adjacentnucleic acid sequence. As used herein, a promoter may include necessarynucleic acid sequences near the start site of transcription. A promoteralso optionally includes distal enhancer or repressor elements, whichcan be located as much as several thousand base pairs from the startsite of transcription. A promoter also optionally includes one or morecopies of an upstream activation sequence (UAS).

“Recombinant” refers to a cell, nucleic acid, protein, or vector, whichhas been modified due to introduction of an exogenous nucleic acid oralteration of a native nucleic acid. Resulting cells, nucleic acids,proteins or vectors are considered recombinant, as are progeny,offspring, duplications or replications of these are also consideredrecombinant. Thus, e.g., recombinant cells can express genes that arenot found within the native (non-recombinant) form of the cell orexpress native genes differently than those genes are expressed by anon-recombinant cell. Recombinant cells can, without limitation, includerecombinant nucleic acids that encode for a gene product or forsuppression elements such as mutations, knockouts, antisense,interfering RNA (RNAi), or dsRNA that reduce the levels of active geneproduct in a cell. A “recombinant nucleic acid” is derived from nucleicacid originally formed in vitro, in general, by the manipulation ofnucleic acid, e.g., using polymerases, ligases, exonucleases, andendonucleases, or otherwise is in a form not normally found in nature.Recombinant nucleic acids may be produced, for example, to place two ormore nucleic acids in operable linkage Thus, an isolated nucleic acid oran expression vector formed in vitro by ligating DNA molecules that arenot normally joined in nature, are both considered recombinant for thepurposes of this disclosure. Once a recombinant nucleic acid is made andintroduced into a host cell or organism, it may replicate using the invivo cellular machinery of the host cell; however, such nucleic acids,once produced recombinantly, although subsequently replicatedintracellularly, are still considered recombinant for purposes of thisdisclosure. Additionally, a recombinant nucleic acid refers tonucleotide sequences that comprise an endogenous nucleotide sequence andan exogenous nucleotide sequence; thus, an endogenous gene that hasundergone recombination with an exogenous promoter is a recombinantnucleic acid. A “recombinant protein” is a protein made usingrecombinant techniques, i.e., through the expression of a recombinantnucleic acid.

The term “regulatory region” refers to nucleotide sequences that affectthe transcription or translation of a gene but do not encode an aminoacid sequence. Regulatory regions include promoters, operators,enhancers, and silencers.

The term “subsequence” refers to a consecutive nucleotide sequence foundwithin a nucleotide sequence that is less than the full-lengthnucleotide sequence. For example, a subsequence may consist of 100consecutive nucleotides selected from the nucleotide sequence set forthin SEQ ID NO: 1-7. As used herein, a subsequence consists of at leastfifty nucleotides.

“Transformation” refers to the transfer of a nucleic acid into a hostorganism or the genome of a host organism. Host organisms (and theirprogeny) containing the transformed nucleic acid fragments are referredto as “recombinant”, “transgenic” or “transformed” organisms. Thus,isolated polynucleotides of the present disclosure can be incorporatedinto recombinant constructs, typically DNA constructs, capable ofintroduction into and replication in a host cell. Such a construct canbe a vector that includes a replication system and sequences that arecapable of transcription and translation of a polypeptide-encodingsequence in a given host cell. Typically, expression vectors include,for example, one or more cloned genes under the transcriptional controlof 5′ and 3′ regulatory sequences and a selectable marker. Such vectorsalso can contain a promoter regulatory region (e.g., a regulatory regioncontrolling inducible or constitutive, environmentally- ordevelopmentally-regulated, or location-specific expression), atranscription initiation start site, a ribosome binding site, atranscription termination site, and/or a polyadenylation signal.Alternatively, a cell may be transformed with a single genetic element,such as a promoter, which may result in genetically stable inheritanceupon integrating into the host organism's genome, such as by homologousrecombination.

The term “transformed cell” refers to a cell that has undergone atransformation. Thus, a transformed cell comprises the parent's genomeand an inheritable genetic modification. Embodiments include progeny andoffspring of such transformed cells.

The term “substantially the same” or “not significantly different”refers to a level of expression that is not significantly different thanwhat it is compared to. Alternatively, or in conjunction, the termsubstantially the same refers to a level of expression that is less than2, 1.5, or 1.25 fold different than the expression or activity level itis compared to.

The term “promoter region of a Yarrowia gene” or “Yarrowia promoterregion” refers to the 5′ upstream untranslated region in front of the‘ATG’ translation initiation codon of a Yarrowia gene, or sequencesderived therefrom, and that is necessary for expression of connectedsequence. Thus, it is believed that promoter regions of a Yarrowia genewill comprise a portion of the −1000 bp 5′ upstream of a Yarrowia gene.The sequence of the Yarrowia promoter region may correspond exactly tonative sequence upstream of the Yarrowia gene (i.e., a “wildtype” or“native” Yarrowia promoter); alternately, the sequence of the Yarrowiapromoter region may be “modified” or “mutated”, thereby comprisingvarious substitutions, deletions, and/or insertions of one or morenucleotides relative to a wildtype or native Yarrowia promoter. Thesemodifications can result in a modified Yarrowia promoter havingincreased, decreased or equivalent promoter activity, when compared tothe promoter activity of the corresponding wildtype or native Yarrowiapromoter. The term “mutant promoter” or “modified promoter” willencompass natural variants and in vitro generated variants obtainedusing methods well known in the art (e.g., classical mutagenesis,site-directed mutagenesis and “DNA shuffling”).

As used herein, the term “complementary” and derivatives thereof areused in reference to pairing of nucleic acids by the well-known rulesthat A pairs with T or U and C pairs with G. Complement can be “partial”or “complete”. In partial complement, only some of the nucleotides arematched according to the base pairing rules; while in complete or totalcomplement, all the bases are matched according to the pairing rule. Thedegree of complementarity between the nucleic acid strands may havesignificant an effect on the efficiency and strength of hybridizationbetween two nucleic acid strands as is well known in the art. Theefficiency and strength of hybridization depends upon the detectionmethod.

As used herein, the terms “or” and “and/or” are utilized to describemultiple components in combination or exclusive of one another. Forexample, “x, y, and/or z” can refer to “x” alone, “y” alone, “z” alone,“x, y, and z,” “(x and y) or z,” “x or (y and z),” or “x or y or z.” Itis specifically contemplated that x, y, or z may be specificallyexcluded from an embodiment.

Throughout this application, the term “about” is used according to itsplain and ordinary meaning in the area of cell biology to indicate thata value includes the standard deviation of error for the device ormethod being employed to determine the value.

The term “comprising,” which is synonymous with “including,”“containing,” or “characterized by,” is inclusive or open-ended and doesnot exclude additional, unrecited elements or method steps. The phrase“consisting of” excludes any element, step, or ingredient not specified.The phrase “consisting essentially of” limits the scope of describedsubject matter to the specified materials or steps and those that do notmaterially affect its basic and novel characteristics. It iscontemplated that embodiments described in the context of the term“comprising” may also be implemented in the context of the term“consisting of” or “consisting essentially of.”

It is specifically contemplated that any limitation discussed withrespect to one embodiment of the invention may apply to any otherembodiment of the invention. Furthermore, any composition of theinvention may be used in any method of the invention, and any method ofthe invention may be used to produce or to utilize any composition ofthe invention. Aspects of an embodiment set forth in the Examples arealso embodiments that may be implemented in the context of embodimentsdiscussed elsewhere in a different Example or elsewhere in theapplication, such as in the Summary of Invention, Detailed Descriptionof the Embodiments, Claims, and description of Figure Legends.

The use of the word “a” or “an” when used in conjunction with the term“comprising” in the claims and/or the specification may mean “one,” butit is also consistent with the meaning of “one or more,” “at least one,”and “one or more than one.”

II. Microbe Engineering

A. Overview

Exogenous promoters and promoter regions such as any derived from SEQ IDNO:1-7 (e.g., sequences comprising or derived from SEQ ID NO: 1, 2, 5,6, or 7) may be introduced into many different host cells. Suitable hostcells are microbial hosts that can be found broadly within the fungalfamilies. Examples of suitable host strains include but are not limitedto fungal or yeast species, such as Arxula, Aspergillus,Aurantiochytrium, Candida, Claviceps, Cryptococcus, Cunninghamella,Hansenula, Kluyveromyces, Leucosporidiella, Lipomyces, Mortierella,Ogataea, Pichia, Prototheca, Rhizopus, Rhodosporidium, Rhodotorula,Saccharomyces, Schizosaccharomyces, Tremella, Trichosporon, andYarrowia. Yarrowia lipolytica is well-suited for use as the hostmicroorganism because they can accumulate a large percentage of theirweight as triacylglycerols.

Microbial expression systems and expression vectors are well known tothose skilled in the art. Any such expression vector could be used tointroduce the instant promoters into an organism. The promoters may beintroduced into appropriate microorganisms via transformation techniquesto direct the expression of an operably-linked gene. For example, apromoter can be cloned in a suitable plasmid, and a parent cell can betransformed with the resulting plasmid. This approach can be used todrive the expression of a gene that is either operably linked to thepromoter or that becomes operably linked to the promoter following thetransformation event. The plasmid is not particularly limited so long asit renders a desired promoter inheritable to the microorganism'sprogeny.

Vectors or cassettes useful for the transformation of suitable hostcells are well known in the art. Typically the vector or cassettecontains a gene, sequences directing transcription and translation of arelevant gene including the promoter, a selectable marker, and sequencesallowing autonomous replication or chromosomal integration. Suitablevectors comprise a region 5′ of the gene harboring the promoter andother transcriptional initiation controls and a region 3′ of the DNAfragment which controls transcriptional termination. It is preferredwhen both control regions are derived from genes homologous to thetransformed host cell or from closely related species, although it is tobe understood that such control regions need not be derived from thegenes native to the specific species chosen as a production host. Forexample, a Yarrowia lipolytica promoter may be used to drive expressionin other species of yeast including other oleaginous yeasts.

Promoters, cDNAs, and 3′UTRs, as well as other elements of the vectors,can be generated through cloning techniques using fragments isolatedfrom native sources (Green & Sambrook, Molecular Cloning: A LaboratoryManual, (4th ed., 2012); U.S. Pat. No. 4,683,202; incorporated byreference). Alternatively, elements can be generated synthetically usingknown methods (Gene 164:49-53 (1995)).

B. Promoter Sequences

Described herein, in some embodiments, are promoter sequences comprisingSEQ ID NO:1-7, or subsequences or variants thereof, from Yarrowialipolytica that are useful for modulating lipid production. In someembodiments, described herein are promoter sequences comprising SEQ IDNO: 1, 2, 5, 6, or 7, or subsequences or variants thereof. In someembodiments, promoters described herein facilitate reduced expression oractivity of a sequence or gene of interest during a lipid accumulationphase of Yarrowia lipolytica relative to a growth phase. The use of suchpromoters is advantageous when there is a need to eliminate or reduceprotein accumulation during the lipid production phase of an oleaginoushost cell, while maintaining some level of protein activity during thegrowth of the cell. This differential expression of genes during thegrowth cycle can be used to optimize lipid yield or lipid composition. Agene, or a sequence of interest such as a coding sequence is placedunder the control of a promoter to allow a given level of transcriptionand/or activity of the coding sequence during the active growth phase ofa host cell and reduced transcription and/or activity of the codingsequence during a lipid accumulation phase (e.g., a phase comprisinglittle to no nitrogen in a culture). Promoters may include the entirelength of any of SEQ ID NOS:1-7 or sequences having percent identity toany of SEQ ID NOS:1-7 or may be recombinantly engineered to includetranscriptional regulatory regions and/or promoter elements from SEQ IDNOS:1-7 thereby constituting functional variants of SEQ ID NOS:1-7.

Some embodiments relate to nucleic acid sequences comprising SEQ ID NO:1-7. Some embodiments also relate to nucleic subsequences derived fromSEQ ID NOS:1-7. The nucleic acid sequences or promoters disclosed hereinmay comprise conservative substitutions, deletions, and/or insertionswhile still functioning to drive transcription. Thus, a promotersequence may comprise a nucleotide sequence that is at least or at most70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%,84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or99.9% (or any derivable range within) identical to any of SEQ ID NO:1-7, wherein the sequence retains the promoter function and is capableof driving the expression of a coding sequence. A promoter sequence maycomprise a nucleotide sequence that is 70%, 71%, 72%, 73%, 74%, 75%,76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%,99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% (or any derivable rangewithin) identical to any of SEQ ID NO: 1-7, wherein the sequence retainsthe promoter function and is capable of driving the expression of acoding sequence

To determine the percent identity of two nucleotide sequences, thesequences can be aligned for optimal comparison purposes (e.g., gaps canbe introduced in one or both of a first and a second nucleotide sequencefor optimal alignment and non-identical sequences can be disregarded forcomparison purposes). The nucleotides at corresponding nucleotidepositions can then be compared. When a position in the first sequence isoccupied by the same nucleotide as the corresponding position in thesecond sequence, then the molecules are identical at that position. Thepercent identity between the two sequences is a function of the numberof identical positions shared by the sequences, taking into account thenumber of gaps, and the length of each gap, which need to be introducedfor the optimal alignment of the two sequences.

The comparison of sequences and determination of percent identitybetween two sequences can be accomplished using a mathematicalalgorithm. Exemplary computer programs which can be used to determineidentity between two nucleotide sequences include, but are not limitedto, the suite of BLAST programs, e.g., BLASTN, MEGABLAST, and Clustalprograms, e.g., ClustalW, ClustalX, and Clustal Omega.

Sequence searches are typically carried out using the BLASTN program,when evaluating a given nucleotide sequence relative to nucleotidesequences in the GenBank DNA Sequences and other public databases. Analignment of selected sequences in order to determine “% identity”between two or more sequences is performed using for example, theCLUSTAL-W program.

The abbreviation used throughout the specification to refer to nucleicacids comprising and/or consisting of nucleotide sequences are theconventional one-letter abbreviations. Thus when included in a nucleicacid, the naturally occurring encoding nucleotides are abbreviated asfollows: adenine (A), guanine (G), cytosine (C), thymine (T) and uracil(U). Also, the nucleotide sequences presented herein is the 5′→3′direction.

In some cases, the full nucleotide sequence of a promoter is notnecessary to drive transcription, and sequences shorter than thepromoter's full nucleotide sequence can drive transcription of anoperably-linked gene. The minimal portion of a promoter, termed the corepromoter, includes a transcription start site, a binding site for a RNApolymerase, and a binding site for a transcription factor. A promotermay comprise a subsequence of any of SEQ ID NOs: 1-7.

A promoter may comprise a subsequence of SEQ ID NO: 5. A promoter maycomprise a nucleotide sequence that is at least, is, or is at most 70%,71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%,85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% ormore identical to 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62,63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80,81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98,99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112,113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126,127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140,141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154,155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168,169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182,183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196,197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210,211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224,225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238,239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252,253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266,267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280,281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294,295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308,309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322,323, 324, 325, 326, 327, 328, 329, or 330 consecutive nucleotides (orany range derivable therein) of SEQ ID NO: 5. Such a subsequence maystart at position 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34,35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52,53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70,71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88,89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104,105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118,119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132,133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146,147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160,161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174,175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188,189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202,203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216,217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230,231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244,245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258,259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272,273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286,287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300,301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314,315, 316, 317, 318, 319, or 320 of SEQ ID NO:5. Such a subsequence maybe 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66,67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84,85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101,102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115,116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129,130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143,144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157,158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171,172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185,186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199,200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213,214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227,228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241,242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255,256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269,270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283,284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297,298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311,312, 313, 314, 315, 316, 317, 318, 319, or 320 nucleotides in length.

A promoter may comprise a nucleotide sequence that is a subsequence ofany of SEQ ID NO: 1, 2, 3, 4, 6, or 7. A promoter may comprise anucleotide sequence that is at least, is, or is at most 70%, 71%, 72%,73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%,87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%,99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or more identicalto 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66,67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84,85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101,102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115,116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129,130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143,144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157,158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171,172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185,186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199,200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213,214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227,228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241,242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255,256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269,270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283,284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297,298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311,312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325,326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339,340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353,354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367,368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381,382, 383, 384, 385, 386, 387, 388, 389, 390, 391, 392, 393, 394, 395,396, 397, 398, 399, 400, 401, 402, 403, 404, 405, 406, 407, 408, 409,410, 411, 412, 413, 414, 415, 416, 417, 418, 419, 420, 421, 422, 423,424, 425, 426, 427, 428, 429, 430, 431, 432, 433, 434, 435, 436, 437,438, 439, 440, 441, 442, 443, 444, 445, 446, 447, 448, 449, 450, 451,452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465,466, 467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478, 479,480, 481, 482, 483, 484, 485, 486, 487, 488, 489, 490, 491, 492, 493,494, 495, 496, 497, 498, 499, 500, 501, 502, 503, 504, 505, 506, 507,508, 509, 510, 511, 512, 513, 514, 515, 516, 517, 518, 519, 520, 521,522, 523, 524, 525, 526, 527, 528, 529, 530, 531, 532, 533, 534, 535,536, 537, 538, 539, 540, 541, 542, 543, 544, 545, 546, 547, 548, 549,550, 600, 700, 800, or 900 consecutive nucleotides (or any rangederivable therein) of SEQ ID NO: 1, 2, 3, 4, 6, or 7. Such a subsequencemay start at position 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33,34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51,52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69,70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87,88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104,105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118,119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132,133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146,147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160,161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174,175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188,189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202,203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216,217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230,231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244,245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258,259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272,273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286,287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300,301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314,315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328,329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342,343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356,357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370,371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, 383, 384,385, 386, 387, 388, 389, 390, 391, 392, 393, 394, 395, 396, 397, 398,399, 400, 401, 402, 403, 404, 405, 406, 407, 408, 409, 410, 411, 412,413, 414, 415, 416, 417, 418, 419, 420, 421, 422, 423, 424, 425, 426,427, 428, 429, 430, 431, 432, 433, 434, 435, 436, 437, 438, 439, 440,441, 442, 443, 444, 445, 446, 447, 448, 449, 450, 451, 452, 453, 454,455, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465, 466, 467, 468,469, 470, 471, 472, 473, 474, 475, 476, 477, 478, 479, 480, 481, 482,483, 484, 485, 486, 487, 488, 489, 490, 491, 492, 493, 494, 495, 496,497, 498, 499, 500, 501, 502, 503, 504, 505, 506, 507, 508, 509, 510,511, 512, 513, 514, 515, 516, 517, 518, 519, 520, 521, 522, 523, 524,525, 526, 527, 528, 529, 530, 531, 532, 533, 534, 535, 536, 537, 538,539, 540, 541, 542, 543, 544, 545, 546, 547, 548, 549, 550, 600, 700,800, or 900 of any of SEQ ID NOs:1, 2, 3, 4, 6, or 7. Such a subsequencemay be 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65,66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83,84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100,101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114,115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128,129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142,143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156,157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170,171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184,185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198,199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212,213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226,227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240,241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254,255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268,269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282,283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296,297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310,311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324,325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338,339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352,353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366,367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380,381, 382, 383, 384, 385, 386, 387, 388, 389, 390, 391, 392, 393, 394,395, 396, 397, 398, 399, 400, 401, 402, 403, 404, 405, 406, 407, 408,409, 410, 411, 412, 413, 414, 415, 416, 417, 418, 419, 420, 421, 422,423, 424, 425, 426, 427, 428, 429, 430, 431, 432, 433, 434, 435, 436,437, 438, 439, 440, 441, 442, 443, 444, 445, 446, 447, 448, 449, 450,451, 452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464,465, 466, 467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478,479, 480, 481, 482, 483, 484, 485, 486, 487, 488, 489, 490, 491, 492,493, 494, 495, 496, 497, 498, 499, 500, 501, 502, 503, 504, 505, 506,507, 508, 509, 510, 511, 512, 513, 514, 515, 516, 517, 518, 519, 520,521, 522, 523, 524, 525, 526, 527, 528, 529, 530, 531, 532, 533, 534,535, 536, 537, 538, 539, 540, 541, 542, 543, 544, 545, 546, 547, 548,549, 550, 600, 700, 800, or 900 nucleotides in length.

The term “subsequence” refers to a consecutive nucleotide sequence foundwithin a nucleotide sequence that is less than the full-lengthnucleotide sequence. For example, a subsequence may consist of 100consecutive nucleotides selected from the nucleotide sequence set forthin SEQ II) NO: 1-7.

Additionally, two promoters may be combined. For example, the region ofa first promoter that binds an RNA polymerase may be combined with aregion of a second promoter that binds one or more transcription factorsto create a hybrid promoter. Thus, a subsequence of a promoter may becombined with another promoter to change the transcription factors thatregulate the transcription of an operably-linked gene. Thus, a promotermay comprise a nucleotide sequence that is at least or is 70%, 71%, 72%,73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%,87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%,99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or more identicalto 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66,67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84,85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 105,110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175,180, 185, 190, 195, 200, 205, 210, 215, 220, 225, 230, 235, 240, 245,250, 255, 260, 265, 270, 275, 280, 285, 290, 295, or 300 consecutivenucleotides found anywhere in SEQ ID NO: 1-7.

Additionally, provided herein is an isolated nucleic acid moleculecomprising a promoter region of Yarrowia from any of: (a) SEQ ID NO:1;(b) SEQ ID NO:2; (c) SEQ ID NO:3; (d) SEQ ID NO:4; (e) SEQ ID NO:5; (f)SEQ ID NO:6, (g) SEQ ID NO:7 wherein the promoter optionally comprisesat least or at most one modification selected from the group consistingof: (i) a deletion at the 3′-terminus and/or the 5′-terminus of 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40,41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58,59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76,77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94,95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109,110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123,124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137,138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151,152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165,166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179,180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193,194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207,208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221,222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235,236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249,250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263,264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277,278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291,292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305,306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319,320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333,334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347,348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361,362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375,376, 377, 378, 379, 380, 381, 382, 383, 384, 385, 386, 387, 388, 389,390, 391, 392, 393, 394, 395, 396, 397, 398, 399, 400, 401, 402, 403,404, 405, 406, 407, 408, 409, 410, 411, 412, 413, 414, 415, 416, 417,418, 419, 420, 421, 422, 423, 424, 425, 426, 427, 428, 429, 430, 431,432, 433, 434, 435, 436, 437, 438, 439, 440, 441, 442, 443, 444, 445,446, 447, 448, 449, 450, 451, 452, 453, 454, 455, 456, 457, 458, 459,460, 461, 462, 463, 464, 465, 466, 467, 468, 469, 470, 471, 472, 473,474, 475, 476, 477, 478, 479, 480, 481, 482, 483, 484, 485, 486, 487,488, 489, 490, 491, 492, 493, 494, 495, 496, 497, 498, 499, 500, 501,502, 503, 504, 505, 506, 507, 508, 509, 510, 511, 512, 513, 514, 515,516, 517, 518, 519, 520, 521, 522, 523, 524, 525, 526, 527, 528, 529,530, 531, 532, 533, 534, 535, 536, 537, 538, 539, 540, 541, 542, 543,544, 545, 546, 547, 548, 549, 550, 551, 552, 553, 554, 555, 556, 557,558, 559, 560, 561, 562, 563, 564, 565, 566, 567, 568, 569, 570, 571,572, 573, 574, 575, 576, 577, 578, 579, 580, 581, 582, 583, 584, 585,586, 587, 588, 589, 590, 591, 592, 593, 594, 595, 596, 597, 598, 599,600, 601, 602, 603, 604, 605, 606, 607, 608, 609, 610, 611, 612, 613,614, 615, 616, 617, 618, 619, 620, 621, 622, 623, 624, 625, 626, 627,628, 629, 630, 631, 632, 633, 634, 635, 636, 637, 638, 639, 640, 641,642, 643, 644, 645, 646, 647, 648, 649, 650, 651, 652, 653, 654, 655,656, 657, 658, 659, 660, 661, 662, 663, 664, 665, 666, 667, 668, 669,670, 671, 672, 673, 674, 675, 676, 677, 678, 679, 680, 681, 682, 683,684, 685, 686, 687, 688, 689, 690, 691, 692, 693, 694, 695, 696, 697,698, 699, 700, 701, 702, 703, 704, 705, 706, 707, 708, 709 or 710consecutive nucleotides.

A promoter may further comprise and/or be linked to an upstreamactivation sequence (UAS). A UAS may be capable of strengthening theability of a promoter sequence to regulate gene expression. A UAS may beconfigured to increase the expression of a coding sequence operablylinked to a promoter sequence (e.g., a sequence comprising any of SEQ IDNOs: 1-7). A promoter may comprise or be linked to 1, 2, 3, 4, 5, 6, 7,8, 9, or 10 copies of a UAS, or more.

C. Vectors and Vector Components

Vectors for the transformation of microorganisms in accordance with thepresent disclosure can be prepared by known techniques familiar to thoseskilled in the art in view of the disclosure herein. A vector typicallycontains one or more genes, in which each gene codes for the expressionof a desired product (the gene product) and is operably linked to one ormore control sequences that regulate gene expression (i.e., a promoter),or the vector targets a gene, control sequence, or other nucleotidesequence to a particular location in the recombinant cell.

In general, microbial expression systems and expression vectorscontaining regulatory sequences that direct high level expression offoreign proteins are well known to those skilled in the art. Any ofthese could be used to construct chimeric genes, which could then beintroduced into appropriate microorganisms via transformation to providehigh-level expression of the encoded enzymes.

Any nucleic acid vector may encode a promoter. A plasmid may be aconvenient vector because plasmids may be manipulated and replicated inbacterial hosts. In some embodiments, a linear DNA molecule may be apreferable vector, for example, to eliminate plasmid nucleotidesequences prior to transformation. Linear DNA may be obtained from therestriction digest of a plasmid or by PCR amplification. PCR may be usedto generate a linear DNA vector by amplifying plasmid DNA, genomic DNA,synthetic DNA, or any other template. For example, PCR may be used togenerate a linear DNA vector from overlapping oligonucleotide fragments.Suitable vectors are not limited to DNA; for example, the RNA of aretroviral vector may be utilized to transform a cell with a desiredpromoter.

The vector may comprise both the promoter and a gene such that thepromoter and gene are operably linked. Alternatively, the vector may bedesigned so that the promoter becomes operably linked to a gene aftertransformation of the parent cell. For example, a first vectorcontaining the promoter may be designed to recombine with a secondvector containing a gene such that successful transformation andrecombination events cause the promoter and gene to become operablylinked in a host cell. Alternatively, a vector containing the promotermay be designed to recombine with a gene in the genome of the host cellor otherwise integrate into the genome of the host cell. In thisembodiment, the exogenous promoter may replace an endogenous promoter.

A vector may comprise one or more additional promoters, genes, and/orother sequences. For example, a vector may comprise a first promotercomprising SEQ ID NO: 1, 2, 5, 6, or 7, or a subsequence or functionalvariant thereof, operably linked to a first gene, and a second promoter(e.g., a constitutive promoter) operably linked to a second gene. Avector may comprise at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 promoters,or more. A vector may comprise at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10genes, or more. A vector comprising multiple promoters linked tomultiple genes may be useful in, for example, reducing the expression ofa first gene while simultaneously increasing the expression of a secondgene.

1. Control Sequences

Control sequences are nucleic acids that regulate the expression of acoding sequence or direct a gene product to a particular location in oroutside a cell. Control sequences that regulate expression include, forexample, promoters that regulate the transcription of a coding sequenceand terminators that terminate the transcription of a coding sequence.Another control sequence is a 3′ untranslated sequence located at theend of a coding sequence that encodes a polyadenylation signal. Controlsequences that direct gene products to particular locations includethose that encode signal peptides, which direct the protein to whichthey are attached to a particular location in or outside the cell.

Thus, an exemplary vector design for the expression of a promoter in amicrobe contains a coding sequence for a desired gene product (forexample, a selectable marker, or an enzyme) in operable linkage with apromoter active in yeast. Alternatively, if the vector does not containa gene in operable linkage with a promoter, the promoter can betransformed into the cells such that it becomes operably linked to anendogenous gene at the point of vector integration.

The promoter used to express a gene can be the promoter naturally linkedto that gene or a different promoter.

The inclusion of a termination region control sequence is optional, andif employed, the choice is primarily one of convenience, as terminationregions are relatively interchangeable. The termination region may benative to the transcriptional initiation region (the promoter), may benative to the DNA sequence of interest, or may be obtainable fromanother source (See, e.g., Chen & Orozco, Nucleic Acids Research 16:8411(1988)).

2. Genes

Typically, a gene includes a promoter, coding sequence, and terminationcontrol sequences. When assembled by recombinant DNA technology, a genemay be termed an expression cassette and may be flanked by restrictionsites for convenient insertion into a vector that is used to introducethe recombinant gene into a host cell. The expression cassette can beflanked by DNA sequences from the genome or other nucleic acid target tofacilitate stable integration of the expression cassette into the genomeby homologous recombination. Alternatively, the vector and itsexpression cassette may remain unintegrated (e.g., an episome), in whichcase, the vector typically includes an origin of replication, which iscapable of providing for replication of the vector DNA.

A common gene present on a vector is a gene that codes for a protein,the expression of which allows the recombinant cell containing theprotein to be differentiated from cells that do not express the protein.Such a gene, and its corresponding gene product, is called a selectablemarker or selection marker. Any of a wide variety of selectable markerscan be employed in a transgene construct useful for transforming theorganisms of the disclosure.

For optimal expression of a recombinant protein, it is beneficial toemploy coding sequences that produce mRNA with codons optimally used bythe host cell to be transformed. Thus, proper expression of transgenescan require that the codon usage of the transgene matches the specificcodon bias of the organism in which the transgene is being expressed.The precise mechanisms underlying this effect are many, but include theproper balancing of available aminoacylated tRNA pools with proteinsbeing synthesized in the cell, coupled with more efficient translationof the transgenic messenger RNA (mRNA) when this need is met. When codonusage in the transgene is not optimized, available tRNA pools are notsufficient to allow for efficient translation of the transgenic mRNAresulting in ribosomal stalling and termination and possible instabilityof the transgenic mRNA.

D. Homologous Recombination

Homologous recombination may be used to substitute one nucleotidesequence with a different nucleotide sequence. Thus, homologousrecombination may be used to substitute all or part of an endogenouspromoter that drives the expression of a gene in an organism with all orpart of an exogenous promoter. Additionally, homologous recombinationmay be used to combine two nucleic acids that contain a homologousnucleotide sequence.

Homologous recombination is the ability of complementary DNA sequencesto align and exchange regions of homology. For example, transgenic DNA(“donor”) containing sequences homologous to the genomic sequences beingtargeted (“template”) may be generated and introduced into an organismto undergo recombination with the organism's genomic sequences.

The ability to carry out homologous recombination in a host organism hasmany practical implications for what can be carried out at the moleculargenetic level and is useful in the generation of microbes that produce adesired product. By its very nature, homologous recombination is aprecise gene targeting event; hence, most transgenic lines generatedwith the same targeting sequence will be essentially identical in termsof phenotype, necessitating the screening of far fewer transformationevents. Homologous recombination also targets gene insertion events intothe host chromosome, potentially resulting in excellent geneticstability, even in the absence of genetic selection.

Because homologous recombination is a precise gene targeting event, itcan be used to precisely modify any nucleotide(s) within a gene orregion of interest, so long as sufficient flanking regions have beenidentified. Therefore, homologous recombination can be used to modifythe regulatory sequences impacting the expression of RNA and/orproteins. It can also modify protein coding regions, for example, bymodifying enzyme activities such as substrate specificity, bindingaffinities and Km, and thus, it may affect a desired change in themetabolism of a host cell. Homologous recombination provides a powerfulmeans to manipulate the host genome resulting in gene targeting, geneconversion, gene deletion, gene duplication, gene inversion andexchanging gene expression regulatory elements such as promoters,enhancers and 3′UTRs. Thus, homologous recombination allows for thesubstitution of an endogenous promoter in an organism with a differentpromoter. An exogenous promoter may provide advantages over theendogenous promoter; for example, the exogenous promoter may increase ordecrease the transcription of an operably-linked gene, or the exogenouspromoter may allow for the regulation of transcription by differentcellular processes relative to the endogenous promoter.

Homologous recombination can be achieved by using targeting constructscontaining pieces of endogenous sequences to “target” the gene or regionof interest within the endogenous host cell genome. Such targetingsequences can be located upstream or downstream of the gene or region ofinterest, or flank the gene/region of interest. Such targetingconstructs can be transformed into the host cell as circular plasmidDNA, optionally including nucleotide sequences from the plasmid;linearized DNA, such as a plasmid restriction digest; PCR product, suchas the amplification of overlapping oligonucleotides; or any other meansof introducing DNA into a cell. In some cases, it may be advantageous tofirst expose the homologous sequences within the transgenic DNA (donorDNA) by cutting the transgenic DNA with a restriction enzyme, which canincrease recombination efficiency and decrease the occurrence ofnon-specific recombination events. Other methods of increasingrecombination efficiency include using PCR to generate transformingtransgenic DNA containing linear ends homologous to the genomicsequences being targeted.

E. Transformation

Cells can be transformed by any suitable technique including, e.g.,biolistics, electroporation, glass bead transformation, and siliconcarbide whisker transformation. Any convenient technique for introducinga transgene into a microorganism can be employed in the presentdisclosure. Transformation can be achieved by, for example, the methodof D. M. Morrison (Methods in Enzymology 68:326 (1979)), the method byincreasing permeability of recipient cells for DNA with calcium chloride(Mandel & Higa, J. Molecular Biology, 53:159 (1970)), or the like.

Examples of the expression of transgenes in oleaginous yeast (e.g.,Yarrowia lipolytica) can be found in the literature (Bordes et al., J.Microbiological Methods, 70:493 (2007); Chen et al., AppliedMicrobiology & Biotechnology 48:232 (1997)).

Vectors for the transformation of microorganisms can be prepared byknown techniques. In one embodiment, an exemplary vector for theexpression of a gene in a microorganism comprises a gene encoding aprotein in operable linkage with a promoter. Alternatively, if thepromoter is not operably linked with the gene of interest, the promotermay be transformed into a cell such that it becomes operably linked to anative gene at the point of vector integration. Additionally, microbesmay be transformed with two vectors simultaneously (See, e.g., Protist155:381-93 (2004)). The transformed cells can be optionally selectedbased upon their ability to grow in the presence of an antibiotic orother selectable marker under conditions in which untransformed cellswould not grow.

F. Promoter Targets

One or more promoters of the present disclosure (e.g., a sequencecomprising SEQ ID NO: 1, 2, 5, 6, or 7, or a subsequence or functionalvariant thereof) may be operably linked to one or more nucleic acidsequences. A promoter may be operably linked to a target. A target maybe a gene. A target may be a regulatory RNA (e.g., siRNA, shRNA, miRNA,lncRNA, piRNA, etc.). Examples of targets which may be linked topromoters described herein for use in the disclosed methods include, forexample, genes involved in fatty acid synthesis (e.g., FAS1/2, ELO1,ELO2, FAD2, OLE1, SCT1, LSC1, PAH1, DGA1, DGA2, LRO1, etc.), genesinvolved in lipid breakdown (e.g. peroxisomal enzymes such as PXA1/2,PEX10; fatty acid oxidation genes such as MFE1; lipases such as TGL3,TGL4; etc.), genes involved in fatty acid transport or activation (e.g.ACB1, FAA1-4, etc.), genes involved in diverting carbon flux toundesirable products, genes involved in synthesis or breakdown ofglucose, and genes involved in energy metabolism (e.g. phosphoglucoseisomerase, phosphofructokinase, ATP citrate lyase, citrate synthaseetc.). A target linked to a promoter may be a Yarrowia lipolytica gene.Alternatively, a target linked to a promoter may not be a Yarrowialipolytica gene.

A promoter may be linked to a target by introducing the promoter and thetarget into a nucleic acid molecule, for example, a vector. A vector maybe introduced into a cell, thereby expressing the promoter and thetarget. In one embodiment, a promoter may be linked to a target byintroducing a promoter into DNA of a cell, for example, via homologousrecombination.

III. Exemplary Nucleic Acids and Methods

Through analysis of publicly available transcriptomic data, transcriptswere identified whose abundance dropped at high carbon-to-nitrogenratios. The promoter regions of these genes were designated asPR104-PR110 (SEQ ID NO:1-7). The inventors took the 1000 bp (or 370 bpfor PR108 (SEQ ID NO:5) where the intergenic region is shorter) upstreamof the transcription start site of each of the corresponding genes andused them to drive transcription of genes of interest in order to obtainactivity during growth but not during lipid accumulation (a transitioninvolving an increase in the carbon-to-nitrogen ratio). In someembodiments, a nucleic acid comprises a nucleotide sequence having atleast 70% identity with SEQ ID NO: 1, 2, 5, 6, or 7, or any subsequenceor functional variant thereof.

In some embodiments, the nucleic acid comprises a nucleotide sequencehaving at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%,81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%,99.7%, 99.8%, 99.9% or more sequence identity (or any range derivabletherein) with the sequence set forth in SEQ ID NO: 1, 2, 5, 6 or 7. Insome embodiments, the nucleic acid comprises a nucleotide sequence thatis 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%,84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%or more sequence identity (or any range derivable therein) with thesequence set forth in SEQ ID NO: 1, 2, 5, 6 or 7. In other embodiments,the nucleic acid comprises a nucleotide sequence having at least 70%,71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%,85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% ormore sequence identity (or any range derivable therein) with asubsequence of SEQ ID NO: 1, 2, 5, 6 or 7. In other embodiments, thenucleic acid comprises a nucleotide sequence having 70%, 71%, 72%, 73%,74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%,88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%,99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or more sequenceidentity (or any range derivable therein) with a subsequence of SEQ IDNO: 1, 2, 5, 6 or 7. In some embodiments, the nucleic acid comprises thenucleotide sequence set forth in SEQ ID NO: 1, 2, 5, 6 or 7. In otherembodiments, the nucleic acid comprises a nucleotide sequence consistingof a subsequence of SEQ ID NO: 1, 2, 5, 6 or 7. In certain embodiments,the subsequence retains promoter activity. In certain embodiments, thesubsequence retains 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%,13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%,27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%,41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%,55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%,69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%,83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, or 99% or 100% (or any range derivable therein) of thepromoter activity of the full-length nucleotide sequence. In certainembodiments, the subsequence retains promoter activity. In certainembodiments, the subsequence retains at least 1%, 2%, 3%, 4%, 5%, 6%,7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%,22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%,36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%,50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%,64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%,78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or 100% (or any rangederivable therein) of the promoter activity of the full-lengthnucleotide sequence. In certain embodiments, the subsequence retains thepromoter activity of the full-length nucleotide sequence.

In some embodiments, the subsequence is 50, 51, 52, 53, 54, 55, 56, 57,58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75,76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93,94, 95, 96, 97, 98, 99, 100, 105, 110, 115, 120, 125, 130, 135, 140,145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 205, 210,215, 220, 225, 230, 235, 240, 245, 250, 255, 260, 265, 270, 275, 280,285, 290, 295, or 300 nucleotides long or longer. In some embodiments,the subsequence comprises 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60,61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78,79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96,97, 98, 99, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155,160, 165, 170, 175, 180, 185, 190, 195, 200, 205, 210, 215, 220, 225,230, 235, 240, 245, 250, 255, 260, 265, 270, 275, 280, 285, 290, 295, or300 consecutive nucleotides found anywhere in SEQ ID NO: 1, 2, 5, 6 or7. In some embodiments, the subsequence comprises 50, 51, 52, 53, 54,55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72,73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90,91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 105, 110, 115, 120, 125, 130,135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200,205, 210, 215, 220, 225, 230, 235, 240, 245, 250, 255, 260, 265, 270,275, 280, 285, 290, 295, or 300 consecutive nucleotides at the3′-terminus of SEQ ID NO: 1, 2, 5, 6 or 7.

In some embodiments, the nucleic acid comprises a nucleotide sequencehaving or having at least or having at most 70%, 71%, 72%, 73%, 74%,75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%,99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100% (or any rangederivable therein) sequence identity with 50, 51, 52, 53, 54, 55, 56,57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74,75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92,93, 94, 95, 96, 97, 98, 99, 100, 105, 110, 115, 120, 125, 130, 135, 140,145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 205, 210,215, 220, 225, 230, 235, 240, 245, 250, 255, 260, 265, 270, 275, 280,285, 290, 295, or 300 consecutive nucleotides found anywhere in SEQ IDNO: 1, 2, 5, 6 or 7. In some embodiments, the nucleic acid comprises anucleotide sequence consisting of 50, 51, 52, 53, 54, 55, 56, 57, 58,59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76,77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94,95, 96, 97, 98, 99, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145,150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 205, 210, 215,220, 225, 230, 235, 240, 245, 250, 255, 260, 265, 270, 275, 280, 285,290, 295, or 300 consecutive nucleotides found anywhere in SEQ ID NO: 1,2, 5, 6 or 7. In certain embodiments, the nucleotide sequence retainspromoter activity. In certain embodiments, the nucleotide sequenceretains at least, at most or retains 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%,10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%,24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%,38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%,52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%,66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%,80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, or 99% or 100% (or any percentage derivabletherein) of the promoter activity of the full-length nucleotidesequence. In certain embodiments, the nucleotide sequence retains thepromoter activity of the full-length nucleotide sequence.

In some embodiments, the nucleic acid comprises a nucleotide sequencehaving at least or having at most or having 70%, 71%, 72%, 73%, 74%,75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%,99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100% (or anypercentage derivable therein) sequence identity with 50, 51, 52, 53, 54,55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72,73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90,91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 105, 110, 115, 120, 125, 130,135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200,205, 210, 215, 220, 225, 230, 235, 240, 245, 250, 255, 260, 265, 270,275, 280, 285, 290, 295, or 300 consecutive nucleotides at the3′-terminus of SEQ ID NO: 1, 2, 5, 6 or 7. In some embodiments, thenucleic acid comprises a nucleotide sequence consisting of 50, 51, 52,53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70,71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88,89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 105, 110, 115, 120,125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190,195, 200, 205, 210, 215, 220, 225, 230, 235, 240, 245, 250, 255, 260,265, 270, 275, 280, 285, 290, 295, or 300 consecutive nucleotides at the3′-terminus of SEQ ID NO: 1, 2, 5, 6 or 7. In certain embodiments, thenucleotide sequence retains promoter activity. In certain embodiments,the nucleotide sequence retains at least or retains 1%, 2%, 3%, 4%, 5%,6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%,21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%,35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%,49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%,63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%,77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or 100% (or anypercentage derivable therein) of the promoter activity of thefull-length nucleotide sequence. In certain embodiments, the nucleotidesequence retains the promoter activity of the full-length nucleotidesequence.

Although promoters PR106 (SEQ ID NO: 3) and PR107 (SEQ ID NO: 4) drovevery low levels of expression compared to promoters PR104 (SEQ ID NO:1), PR105 (SEQ ID NO: 2), PR108 (SEQ ID NO: 5), PR109 (SEQ ID NO: 6),and PR110 (SEQ ID NO: 7), in some embodiments, a nucleic acid comprisesa nucleotide sequence having at least 70% identity with SEQ ID NO: 3 or4, or any subsequence or functional variant thereof.

Vectors Comprising Promoters Derived from Yarrowia lipolytica

Some embodiments relate to a vector comprising a nucleotide sequenceencoding a promoter from Yarrowia lipolytica, wherein the promoter isSEQ ID NO:1, 2, 5, 6 or 7, or a subsequence or functional variantthereof. In some embodiments, the vector is a plasmid. In otherembodiments, the vector is a linear DNA molecule.

In some embodiments, the nucleotide sequence has or has at least 70%,71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%,85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or100% (or any percentage derivable therein) sequence identity with thesequence set forth in SEQ ID NO: 1, 2, 5, 6 or 7. In other embodiments,the nucleotide sequence has or has at least 70%, 71%, 72%, 73%, 74%,75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%,99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100% (or anypercentage derivable therein) sequence identity with a subsequence ofSEQ ID NO: 1, 2, 5, 6 or 7. In some embodiments, the nucleotide sequencecomprises the sequence set forth in SEQ ID NO: 1, 2, 5, 6 or 7. In otherembodiments, the nucleotide sequence comprises a subsequence of SEQ IDNO: 1, 2, 5, 6 or 7. In certain embodiments, the subsequence retainspromoter activity. In certain embodiments, the subsequence retains orretains at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%,14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%,28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%,42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%,56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%,70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%,84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, or 99% or 100% (or any percentage derivable therein) of thepromoter activity of the full-length nucleotide sequence. In certainembodiments, the subsequence retains the promoter activity of thefull-length nucleotide sequence.

In some embodiments, the subsequence is 50, 51, 52, 53, 54, 55, 56, 57,58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75,76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93,94, 95, 96, 97, 98, 99, 100, 105, 110, 115, 120, 125, 130, 135, 140,145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 205, 210,215, 220, 225, 230, 235, 240, 245, 250, 255, 260, 265, 270, 275, 280,285, 290, 295, or 300 nucleotides long or longer. In some embodiments,the subsequence comprises 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60,61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78,79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96,97, 98, 99, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155,160, 165, 170, 175, 180, 185, 190, 195, 200, 205, 210, 215, 220, 225,230, 235, 240, 245, 250, 255, 260, 265, 270, 275, 280, 285, 290, 295, or300 consecutive nucleotides found anywhere in SEQ ID NO: 1, 2, 5, 6 or7. In some embodiments, the subsequence comprises 50, 51, 52, 53, 54,55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72,73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90,91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 105, 110, 115, 120, 125, 130,135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200,205, 210, 215, 220, 225, 230, 235, 240, 245, 250, 255, 260, 265, 270,275, 280, 285, 290, 295, or 300 consecutive nucleotides at the3′-terminus of SEQ ID NO: 1, 2, 5, 6 or 7.

In some embodiments, the nucleotide sequence has or has at least 70%,71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%,85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or100% (or any percentage derivable therein) sequence identity with 50,51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68,69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86,87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 105, 110, 115,120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185,190, 195, 200, 205, 210, 215, 220, 225, 230, 235, 240, 245, 250, 255,260, 265, 270, 275, 280, 285, 290, 295, or 300 consecutive nucleotidesfound anywhere in SEQ ID NO: 1, 2, 5, 6 or 7. In some embodiments, thenucleotide sequence comprises 50, 51, 52, 53, 54, 55, 56, 57, 58, 59,60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77,78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95,96, 97, 98, 99, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150,155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 205, 210, 215, 220,225, 230, 235, 240, 245, 250, 255, 260, 265, 270, 275, 280, 285, 290,295, or 300 consecutive nucleotides found anywhere in SEQ ID NO: 1, 2,5, 6 or 7. In certain embodiments, the nucleotide sequence retainspromoter activity. In certain embodiments, the nucleotide sequenceretains or retains at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%,11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%,25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%,39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%,53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%,67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%,81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, or 99% or 100% (or any percentage derivable therein)of the promoter activity of the full-length nucleotide sequence. Incertain embodiments, the nucleotide sequence retains the promoteractivity of the full-length nucleotide sequence.

In some embodiments, the nucleotide sequence has or has at least 70%,71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%,85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or100% (or any percentage derivable therein) sequence identity with 50,51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68,69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86,87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 105, 110, 115,120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185,190, 195, 200, 205, 210, 215, 220, 225, 230, 235, 240, 245, 250, 255,260, 265, 270, 275, 280, 285, 290, 295, or 300 consecutive nucleotidesat the 3′-terminus of SEQ ID NO: 1, 2, 5, 6 or 7. In some embodiments,the nucleotide sequence comprises 50, 51, 52, 53, 54, 55, 56, 57, 58,59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76,77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94,95, 96, 97, 98, 99, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145,150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 205, 210, 215,220, 225, 230, 235, 240, 245, 250, 255, 260, 265, 270, 275, 280, 285,290, 295, or 300 consecutive nucleotides at the 3′-terminus of SEQ IDNO: 1, 2, 5, 6 or 7. In certain embodiments, the nucleotide sequenceretains promoter activity. In certain embodiments, the nucleotidesequence retains or retains at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%,10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%,24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%,38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%,52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%,66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%,80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, or 99% (or any percentage derivable therein) ofthe promoter activity of the full-length nucleotide sequence. In certainembodiments, the nucleotide sequence retains the promoter activity ofthe full-length nucleotide sequence.

Although promoters PR106 (SEQ ID NO: 3) and PR107 (SEQ ID NO: 4) drovevery low levels of expression compared to promoters PR104 (SEQ ID NO:1), PR105 (SEQ ID NO: 2), PR108 (SEQ ID NO: 5), PR109 (SEQ ID NO: 6),and PR110 (SEQ ID NO: 7), in some embodiments, the nucleotide sequencehas at least 70% identity with SEQ ID NO: 3 or 4, or any subsequence orfunctional variant thereof.

Transformed Cells and Methods of Transforming Cells with PromotersDerived from Yarrowia lipolytica

In certain aspects, the disclosure relates to a transformed cellcomprising a genetic modification, wherein the genetic modification istransformation with a nucleic acid encoding a promoter from Yarrowialipolytica comprising SEQ ID NO: 1, 2, 5, 6 or 7. In some aspects, thedisclosure relates to methods of expressing a gene in a cell comprisingtransforming a parent cell with a nucleic acid encoding a promoter fromYarrowia lipolytica. In some embodiments, the nucleic acid comprises agene, and the gene and the promoter are operably linked. In otherembodiments, the nucleic acid is designed so that the promoter becomesoperably linked to a gene after transformation of the parent cell.

In some embodiments, the nucleic acid comprises a nucleotide sequencehaving or having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%,79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%,99.6%, 99.7%, 99.8%, 99.9% or more sequence identity (or any percentagederivable therein) with the sequence set forth in SEQ ID NO: 1, 2, 5, 6or 7. In other embodiments, the nucleic acid comprises a nucleotidesequence having or having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%,77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%,99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100% sequence identity (or anypercentage derivable therein) with a subsequence of SEQ ID NO: 1, 2, 5,6 or 7. In some embodiments, the nucleic acid comprises the nucleotidesequence set forth in SEQ ID NO: 1, 2, 5, 6 or 7. In other embodiments,the nucleic acid comprises a nucleotide sequence consisting of asubsequence of SEQ ID NO: 1, 2, 5, 6 or 7. In certain embodiments, thesubsequence retains promoter activity. In certain embodiments, thesubsequence retains or retains at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%,9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%,23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%,37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%,51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%,65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%,79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, or 99% (or any percentage derivabletherein) of the promoter activity of the full-length nucleotidesequence. In certain embodiments, the subsequence retains the promoteractivity of the full-length nucleotide sequence.

In some embodiments, the subsequence is 50, 51, 52, 53, 54, 55, 56, 57,58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75,76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93,94, 95, 96, 97, 98, 99, 100, 105, 110, 115, 120, 125, 130, 135, 140,145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 205, 210,215, 220, 225, 230, 235, 240, 245, 250, 255, 260, 265, 270, 275, 280,285, 290, 295, or 300 nucleotides long or longer. In some embodiments,the subsequence comprises 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60,61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78,79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96,97, 98, 99, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155,160, 165, 170, 175, 180, 185, 190, 195, 200, 205, 210, 215, 220, 225,230, 235, 240, 245, 250, 255, 260, 265, 270, 275, 280, 285, 290, 295, or300 consecutive nucleotides found anywhere in SEQ ID NO: 1, 2, 5, 6 or7. In some embodiments, the subsequence comprises 50, 51, 52, 53, 54,55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72,73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90,91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 105, 110, 115, 120, 125, 130,135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200,205, 210, 215, 220, 225, 230, 235, 240, 245, 250, 255, 260, 265, 270,275, 280, 285, 290, 295, or 300 consecutive nucleotides at the3′-terminus of SEQ ID NO: 1, 2, 5, 6 or 7.

In some embodiments, the nucleic acid comprises a nucleotide sequencehaving or having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%,79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%,99.6%, 99.7%, 99.8%, 99.9% or 100% (or any percentage derivable therein)sequence identity with 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61,62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79,80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97,98, 99, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160,165, 170, 175, 180, 185, 190, 195, 200, 205, 210, 215, 220, 225, 230,235, 240, 245, 250, 255, 260, 265, 270, 275, 280, 285, 290, 295, or 300consecutive nucleotides found anywhere in SEQ ID NO: 1, 2, 5, 6 or 7. Insome embodiments, the nucleic acid comprises a nucleotide sequenceconsisting of 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63,64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81,82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99,100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165,170, 175, 180, 185, 190, 195, 200, 205, 210, 215, 220, 225, 230, 235,240, 245, 250, 255, 260, 265, 270, 275, 280, 285, 290, 295, or 300consecutive nucleotides found anywhere in SEQ ID NO: 1, 2, 5, 6 or 7. Incertain embodiments, the nucleotide sequence retains promoter activity.In certain embodiments, the nucleotide sequence retains or retains atleast 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%,16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%,30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%,44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%,58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%,72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%or 100% (or any percentage derivable therein) of the promoter activityof the full-length nucleotide sequence. In certain embodiments, thenucleotide sequence retains the promoter activity of the full-lengthnucleotide sequence.

In some embodiments, the nucleic acid comprises a nucleotide sequencehaving or having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%,79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%,99.6%, 99.7%, 99.8%, 99.9% or more (or any percentage derivable therein)sequence identity with 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61,62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79,80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97,98, 99, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160,165, 170, 175, 180, 185, 190, 195, 200, 205, 210, 215, 220, 225, 230,235, 240, 245, 250, 255, 260, 265, 270, 275, 280, 285, 290, 295, or 300consecutive nucleotides at the 3′-terminus of SEQ ID NO: 1, 2, 5, 6 or7. In some embodiments, the nucleic acid comprises a nucleotide sequenceconsisting of 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63,64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81,82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99,100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165,170, 175, 180, 185, 190, 195, 200, 205, 210, 215, 220, 225, 230, 235,240, 245, 250, 255, 260, 265, 270, 275, 280, 285, 290, 295, or 300consecutive nucleotides at the 3′-terminus of SEQ ID NO: 1, 2, 5, 6 or7. In certain embodiments, the nucleotide sequence retains promoteractivity. In certain embodiments, the nucleotide sequence retains orretains at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%,14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%,28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%,42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%,56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%,70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%,84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, or 99% (or any percentage derivable therein) of the promoteractivity of the full-length nucleotide sequence. In certain embodiments,the nucleotide sequence retains the promoter activity of the full-lengthnucleotide sequence.

Although promoters PR106 (SEQ ID NO: 3) and PR107 (SEQ ID NO: 4) drovevery low levels of expression compared to promoters PR104 (SEQ ID NO:1), PR105 (SEQ ID NO: 2), PR108 (SEQ ID NO: 5), PR109 (SEQ ID NO: 6),and PR110 (SEQ ID NO: 7), in some embodiments, a nucleic acid comprisesa nucleotide sequence having at least 70% identity with SEQ ID NO: 3 or4, or any subsequence or functional variant thereof.

Species of Cells, Parent Cells, and Transformed Host Cells

In some aspects, the current disclosure relates to a host cellcomprising any one of SEQ ID NO:1, 2, 5, 6, or 7, or subsequences orvariants thereof. The host cell may be selected from the groupconsisting of algae, bacteria, molds, fungi, plants, and yeasts. In someembodiments, the cell is selected from the group consisting of Arxula,Aspergillus, Aurantiochytrium, Candida, Claviceps, Cryptococcus,Cunninghamella, Geotrichum, Hansenula, Kluyveromyces, Kodamaea,Leucosporidiella, Lipomyces, Mortierella, Ogataea, Pichia, Prototheca,Rhizopus, Rhodosporidium, Rhodotorula, Saccharomyces,Schizosaccharomyces, Tremella, Trichosporon, Wickerhamomyces, andYarrowia. In certain embodiments, the cell is selected from the groupconsisting of Arxula adeninivorans, Aspergillus niger, Aspergillusorzyae, Aspergillus terreus, Aurantiochytrium limacinum, Candida utilis,Claviceps purpurea, Cryptococcus albidus, Cryptococcus curvatus,Cryptococcus ramirezgomezianus, Cryptococcus terreus, Cryptococcuswieringae, Cunninghamella echinulata, Cunninghamella japonica,Geotrichum fermentans, Hansenula polymorpha, Kluyveromyces lactis,Kluyveromyces marxianus, Kodamaea ohmeri, Leucosporidiella creatinivora,Lipomyces lipofer, Lipomyces starkeyi, Lipomyces tetrasporus,Mortierella isabellina, Mortierella alpina, Ogataea polymorpha, Pichiaciferrii, Pichia guilliermondii, Pichia pastoris, Pichia stipites,Prototheca zopfii, Rhizopus arrhizus, Rhodosporidium babjevae,Rhodosporidium toruloides, Rhodosporidium paludigenum, Rhodotorulaglutinis, Rhodotorula mucilaginosa, Saccharomyces cerevisiae,Schizosaccharomyces pombe, Tremella enchepala, Trichosporon cutaneum,Trichosporon fermentans, Wickerhamomyces ciferrii, and Yarrowialipolytica. Thus, the cell may be Yarrowia lipolytica. The cell may beArxula adeninivorans.

Although promoters PR106 (SEQ ID NO: 3) and PR107 (SEQ ID NO: 4) drovevery low levels of expression compared to promoters PR104 (SEQ ID NO:1), PR105 (SEQ ID NO: 2), PR108 (SEQ ID NO: 5), PR109 (SEQ ID NO: 6),and PR110 (SEQ ID NO: 7), in some embodiments the current disclosurerelates to a host cell comprising any one SEQ ID NO: 3 or 4, orsubsequences or variants thereof.

Growth of Y. lipolytica

The present disclosure also concerns methods for modulating orincreasing the lipid content of a transgenic Yarrowia species thatcomprises operably linking a coding sequence to a nucleotide sequencethat is at least 80% identical to any of SEQ ID NO:1, 2, 5, 6, or 7, ora variant or subsequence thereof such that the coding sequence hasreduced expression in the lipid accumulation phase relative to thegrowth phase.

The transgenic Yarrowia species of the present disclosure can be grownunder conditions that produce the greatest and the most economical yieldof one or more products of interest (e.g., polyunsaturated fatty acids,lipids, etc.). In general, media conditions may be optimized bymodifying the type and amount of carbon source, the type and amount ofnitrogen source, the carbon-to-nitrogen ratio, the amount of differentmineral ions, the oxygen level, growth temperature, pH, length of thebiomass production phase, length of the oil accumulation phase and thetime and method of cell harvest. For example, Yarrowia lipolytica may begrown in a complex media such as yeast extract-peptone-dextrose broth[“YPD”] or a defined minimal media that lacks a component necessary forgrowth and thereby forces selection of the desired expression cassettes(e.g., Yeast Nitrogen Base (DIFCO Laboratories, Detroit, Mich.)).

Fermentation media for the methods and host cells described herein mustcontain a suitable carbon source, such as are described in U.S. Pat. No.7,238,482 and U.S. Pat. Appl. Publ. No. 2011-0059204-A1. Although it iscontemplated that the source of carbon utilized in the presentdisclosure may encompass a wide variety of carbon-containing sources,preferred carbon sources may include sugars (e.g., glucose, invertsucrose, fructose and combinations of thereof), glycerols and/or fattyacids (e.g., those containing between 10-22 carbons).

Nitrogen may be supplied from an inorganic (e.g., (NH.₄)₂SO₄) or organic(e.g., urea or glutamate) source. In addition to appropriate carbon andnitrogen sources, the fermentation media must also contain suitableminerals, salts, cofactors, buffers, vitamins and other components knownto those skilled in the art suitable for the growth of the recombinantmicrobial host cell and the promotion of the enzymatic pathways for EPAproduction. Particular attention is given to several metal ions, such asFe.⁺², Cu⁺², Mn⁺², Co⁺², Zn⁺² and Mg⁺², that promote synthesis of lipidsand PUFAs (Nakahara et al., Ind. Appl. Single Cell Oils, D. J. Kyle andR. Colin, eds. pp 61-97 (1992)).

Growth media for the methods and host cells described herein may becommon commercially prepared media, such as Yeast Nitrogen Base or cornsteep liquors. Other defined or synthetic growth media may also be used.A suitable pH range for the fermentation is typically between about pH4.0 to pH 8.0, wherein pH 5.5 to pH 7.5 is preferred as the range forthe initial growth conditions. The fermentation may be conducted underaerobic or anaerobic conditions, wherein microaerobic conditions arepreferred.

U.S. Pat. Appl. Publ. No. 2009-0093543-A1 provides a detaileddescription of parameters required for a 2-L fermentation of therecombinant Yarrowia lipolytica strain Y4305 (whose maximum productionwas 12.1 EPA % DCW [55.6 EPA % TFAs, with a ratio of EPA % TFAs to LA %TFAs of 3.03] over a period of 162 hours). This disclosure includes adescription of means to prepare inocula from frozen cultures to generatea seed culture, initially culture the yeast under conditions thatpromoted rapid growth to a high cell density, and then culture the yeastto promote lipid and PUFA accumulation (via starving for nitrogen andcontinuously feeding glucose). Process variables including temperature(controlled between 30-32° C.), pH (controlled between 5-7), dissolvedoxygen concentration and glucose concentration were monitored andcontrolled per standard operating conditions. Additional example methodsfor culturing Yarrowia lipolytica for lipid accumulation are describedin Friedlander et al., Biotechnology for Biofuels, 9:77, (2016).

Modified Promoter Regions

Some embodiments are directed to wildtype Yarrowia promoter regions ofSEQ ID NO:1, 2, 6, or 7. Some embodiments are directed to a modifiedYarrowia promoter region relative to SEQ ID NO:1, 2, 6, or 7. A modifiedYarrowia promoter region may comprise the promoter region wherein thepromoter optionally comprises at least one modification selected fromthe group consisting of: a) a deletion at the 5′-terminus of 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41,42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59,60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77,78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95,96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110,111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124,125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138,139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152,153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166,167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180,181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194,195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208,209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222,223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236,237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250,251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264,265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278,279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292,293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306,307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320,321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334,335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348,349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362,363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376,377, 378, 379, 380, 381, 382, 383, 384, 385, 386, 387, 388, 389, 390,391, 392, 393, 394, 395, 396, 397, 398, 399, 400, 401, 402, 403, 404,405, 406, 407, 408, 409, 410, 411, 412, 413, 414, 415, 416, 417, 418,419, 420, 421, 422, 423, 424, 425, 426, 427, 428, 429, 430, 431, 432,433, 434, 435, 436, 437, 438, 439, 440, 441, 442, 443, 444, 445, 446,447, 448, 449, 450, 451, 452, 453, 454, 455, 456, 457, 458, 459, 460,461, 462, 463, 464, 465, 466, 467, 468, 469, 470, 471, 472, 473, 474,475, 476, 477, 478, 479, 480, 481, 482, 483, 484, 485, 486, 487, 488,489, 490, 491, 492, 493, 494, 495, 496, 497, 498, 499, 500, 501, 502,503, 504, 505, 506, 507, 508, 509, 510, 511, 512, 513, 514, 515, 516,517, 518, 519, 520, 521, 522, 523, 524, 525, 526, 527, 528, 529, 530,531, 532, 533, 534, 535, 536, 537, 538, 539, 540, 541, 542, 543, 544,545, 546, 547, 548, 549, 550, 551, 552, 553, 554, 555, 556, 557, 558,559, 560, 561, 562, 563, 564, 565, 566, 567, 568, 569, 570, 571, 572,573, 574, 575, 576, 577, 578, 579, 580, 581, 582, 583, 584, 585, 586,587, 588, 589, 590, 591, 592, 593, 594, 595, 596, 597, 598, 599, 600,601, 602, 603, 604, 605, 606, 607, 608, 609, 610, 611, 612, 613, 614,615, 616, 617, 618, 619, 620, 621, 622, 623, 624, 625, 626, 627, 628,629, 630, 631, 632, 633, 634 or 635 consecutive nucleotides; and b) adeletion at the 3′-terminus of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30,31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48,49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66,67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84,85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101,102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115,116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129,130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143,144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157,158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171,172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185,186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199,200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213,214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227,228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241,242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255,256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269,270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283,284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297,298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311,312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325,326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339,340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353,354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367,368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381,382, 383, 384, 385, 386, 387, 388, 389, 390, 391, 392, 393, 394, 395,396, 397, 398, 399, 400, 401, 402, 403, 404, 405, 406, 407, 408, 409,410, 411, 412, 413, 414, 415, 416, 417, 418, 419, 420, 421, 422, 423,424, 425, 426, 427, 428, 429, 430, 431, 432, 433, 434, 435, 436, 437,438, 439, 440, 441, 442, 443, 444, 445, 446, 447, 448, 449, 450, 451,452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465,466, 467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478, 479,480, 481, 482, 483, 484, 485, 486, 487, 488, 489, 490, 491, 492, 493,494, 495, 496, 497, 498, 499, 500, 501, 502, 503, 504, 505, 506, 507,508, 509, 510, 511, 512, 513, 514, 515, 516, 517, 518, 519, 520, 521,522, 523, 524, 525, 526, 527, 528, 529, 530, 531, 532, 533, 534, 535,536, 537, 538, 539, 540, 541, 542, 543, 544, 545, 546, 547, 548, 549,550, 551, 552, 553, 554, 555, 556, 557, 558, 559, 560, 561, 562, 563,564, 565, 566, 567, 568, 569, 570, 571, 572, 573, 574, 575, 576, 577,578, 579, 580, 581, 582, 583, 584, 585, 586, 587, 588, 589, 590, 591,592, 593, 594, 595, 596, 597, 598, 599, 600, 601, 602, 603, 604, 605,606, 607, 608, 609, 610, 611, 612, 613, 614, 615, 616, 617, 618, 619,620, 621, 622, 623, 624, 625, 626, 627, 628, 629, 630, 631, 632, 633,634 or 635 consecutive nucleotides. A modified Yarrowia promoter regionmay comprise both a deletion from the 5′ terminus and a deletion fromthe 3′ terminus. A modified Yarrowia promoter region disclosed herein,in some embodiments, comprises any contiguous region of SEQ ID NO: 1, 2,6, or 7 capable of acting as a promoter.

Similarly, a modified Yarrowia promoter region may comprise the promoterregion of SEQ ID NO: 5, wherein the promoter optionally comprises atleast one modification selected from the group consisting of: a) adeletion at the 5′-terminus of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30,31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48,49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66,67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84,85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101,102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115,116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129,130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143,144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157,158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171,172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185,186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, or200 consecutive nucleotides; and b) a deletion at the 3′-terminus of 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39,40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57,58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75,76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93,94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108,109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122,123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136,137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150,151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164,165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178,179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192,193, 194, 195, 196, 197, 198, 199, or 200 consecutive nucleotides. Amodified Yarrowia promoter region may comprise both a deletion from the5′ terminus and a deletion from the 3′ terminus. A modified Yarrowiapromoter region disclosed herein, in some embodiments, comprises anycontiguous region of SEQ ID NO: 5 capable of acting as a promoter.

IV. Examples

The following examples are included to demonstrate certain embodimentsof the disclosure. It should be appreciated by those of skill in the artthat the techniques disclosed in the examples which follow representtechniques discovered by the inventor to function well in the practiceof the disclosure. However, those of skill in the art should, in lightof the present disclosure, appreciate that many changes can be made inthe specific embodiments which are disclosed and still obtain a like orsimilar result without departing from the spirit and scope of thedisclosure. The Examples should not be construed as limiting in any way.The contents of all cited references (including literature references,issued patents, published patent applications, and GenBank Accessionnumbers as cited throughout this application) are hereby expresslyincorporated by reference. When definitions of terms in documents thatare incorporated by reference herein conflict with those used herein,the definitions used herein govern.

Example 1

A. Driving Delta-12 Fatty Acid Desaturase (FAD2) with PromotersPR104-PR110 in a Δfad2 Strain

In Y. lipolytica, delta-12 fatty acid desaturase enzyme (FAD2,YALIOB10153) is known to act on oleate (C18:1) and add a double bond inthe Δ12 position to produce linoleate (C18:2). This conversion occurs inthe phospholipid species of oleate and linoleate is naturally producedduring growth. When wild-type cells enter lipid accumulation whichinvolves increased fatty acyl-CoA storage into triacylglycerides (TAGs),there is a natural dilution effect of linoleate in the total fraction offatty acids, although a significant amount of linoleate is still storedas TAGs (FIGS. 8A and B, compare YB-392 16 h and 96 h).

To characterize the promoters in this study, FAD2 was expressed anddriven by PR104-PR110 in a Δ12 desaturase null strain. The endogenousFAD2 was deleted by targeted integration in wild-type strain YB-392 tocreate strain NS419. Tsakraklides et al. demonstrated that deleting thisgene leads to elimination of all detectable linoleate and a concomitantincrease in oleate (Tsakraklides et al., Biotechnology for Biofuels,2018). The Δfad2 strain NS419 was transformed with cassettes drivingexpression of FAD2 from each promoter. All characterizations of thestrains were performed in batch fermentation for 96 hours in lipidproduction media (C:N˜86). These conditions allow for growth during thefirst day while nitrogen is available to produce biomass and lipidaccumulates through the next three days when nitrogen is depleted.Analyses of the cells included gas chromatography (GC) for measuring thelipid composition, qPCR and microscopic imaging. NS419 (Δfad2) andYB-392 (wild-type) were included as controls. 12 transformants werechosen per promoter transformation and grown in 96-deep well plates.Cell pellets were analyzed by GC for lipid composition at day 1 and day4. Two transformants which represented the average performance of the 12transformants were chosen to be re-analyzed in 24 well plates. From the24-well plates, one strain from each set was chosen to be tested inshake flasks along with the control strains NS419 (Δfad2) and YB-392.

Shake flask culture samples were taken at 16 h and 96 h and relativefatty acid composition was determined (FIGS. 8A and 8B). As the Δfad2parent strain NS419 cannot produce linoleate, the levels of linoleate inthe transformed strains can be interpreted as an indication of Δ12desaturase levels and therefore promoter activity in driving FAD2transcription. Based on the percent linoleate present at 16 h (FIG. 8B),transformants with PR110-FAD2, PR105-FAD2 and PR104-FAD2 were thehighest expressors of FAD2 in the growth phase, followed by PR108-FAD2,PR109-FAD2 and finally, PR106-FAD2 and PR107-FAD2 which were similar toNS419 (showing almost no activity during growth and lipid phase). By 96h, all the transformants record linoleate fractions lower than YB-392(wild type strain), even in the case of PR105 and PR110 that containedhigher linoleate fractions than YB-392 at 16 h. Microscopic observationof the transformants and the control strains at 96 h (FIG. 8C) revealsno discernible defects in growth or lipid production and all strainsappear similar to the control strains.

The same samples were analyzed by qPCR to characterize promoter activityand relate transcription to lipid composition (Table 2). The promoterwith the highest FAD2 transcript levels at 16 h (PR110) recorded thehighest percent linoleate, while the promoters with the lowest recorded(PR107) or undetectable FAD2 mRNA levels (PR106 and NS419) had nomeasurable linoleate (Table 2, column B and FIG. 8B). To comparepromoter activity between growth and lipid accumulation, the ratio oftranscript levels and the ratio of linoleate levels at 16 h over 96 hwere calculated (Table 2). FAD2 expression from the native promoter wasrelatively stable between 16 h and 96 h but linoleate content dropped4.5-fold during lipid accumulation in the wild-type strain as fattyacids were directed to TAG storage. Promoters PR105, 108, 109 and 110show repression ranging from 2.6- to >1800-fold between growth and lipidaccumulation (Table 2). This transcriptional repression correlates withthe sharper decrease of percent linoleate for these four promotersbetween the two time points (ranging from 6- to 18-fold) when comparedto YB-392 (FIG. 8B). Note that all strains attained similar accumulationof lipid (FIG. 8C), suggesting that, despite the dilution effect causedby lipid production, FAD2 repression in PR105-110 strains furtherdiminished desaturase activity which was reflected in the lowerlinoleate composition measured. The relative weakness of promotersPR104, PR108 and PR109 in comparison to the native promoter duringgrowth led to lower-than-wild-type linoleate levels at 16 h (Table 2 andFIG. 8B) and contributed to the low linoleate levels during lipidaccumulation

TABLE 2 FAD2 transcript levels of transformants and control strainsmeasured by qPCR. All calculations were carried out using 2-ΔΔCt method(24). FAD2 transcript FAD2 transcript Ratio (FAD2 Ratio relative to YB-relative to YB- transcript at (linoleate at Strain 392 at 16 h 392 at 96h 16 h/96 h) 16 h/96 h) PR104   13%   15%   0.9  8 PR105  109%  0.7% 178 15 PR106 N/A N/A N/A ND PR107 0.25%  0.9%   0.3 ND PR108   14% 1.7%   8.8 14 PR109  7.4%   3%   2.6  7 PR110  231% 0.14% 1855 18 NS419N/A N/A N/A N/A (Δfad2) YB-392  100%  100%   1.1  4.5 (wt) N/A refers toquantities that were too low for detection by qPCR.

B. Driving Delta-9 Fatty Acid Desaturase (OLE1) with PromotersPR104-PR110 in a Δole1 Strain

Y. lipolytica Δ9 desaturase (OLE1, YALI0C05951) acts on saturated fattyacids palmitic acid (C16:0) and stearic acid (C18:0) to produce thecorresponding unsaturated fatty acids palmitoleic acid (C16:1) and oleicacid (C18:1). Deletion of the Y. lipolytica Δ9 desaturase OLE1 causesauxotrophy for monounsaturated fatty acids and reintroduction of anactive Δ9 desaturase rescues growth on unsupplemented media.

Similar to the above experiment, a desaturase null strain was used. OLE1was deleted by targeted integration in wild-type strain YB-392 to createstrain NS418. NS418 was cultured in media supplemented withmonounsaturated fatty acids to maintain viability and transformed withexpression cassettes for containing OLE1 driven by PR104-110.Transformants were selected through antibiotic resistance onsupplemented plates and then tested for their ability to rescue growthon unsupplemented media. Promoters PR104, PR105, PR109 and PR110 enabledgrowth on media without supplementation, indicating that sufficient Δ9desaturase activity was produced during growth phase to supply essentialmonounsaturated fatty acids. PR106-PR108 strains, similar to the parentΔole1 strain NS418, could not grow without supplementation suggestingthat OLE1 expression was below levels necessary for growth. Thesestrains were therefore excluded from subsequent analysis as they cannotgrow in the lipid production media which does not containmonounsaturated fatty acids.

Representative transformants of PR104, PR105, PR109, PR110-OLE1 andYB-392 were sampled, and analyzed as for the FAD2 experiment.Composition measurements (FIG. 9A) revealed varying lipid profiles forPR104, PR105, PR109 and PR110 transformants compared to each other andcompared to wild type strain. To better understand the OLE1 expressioneffect, the direct (C16:1, C18:1) and indirect products (C18:2, madefrom C18:1) of Δ9 desaturase were combined and reported in FIG. 9B. Thereduction of Δ9 desaturated fatty acids by 96 h compared to the wildtype strain suggests reduced OLE1 activity after growth. Microscopicobservation of the transformants at 96 h reveal very small lipid bodieswhen compared to YB-392 (FIG. 9C). OLE1 is known to be an important genein the lipid production pathway. Low OLE1 levels during the lipidaccumulation phase may result in strains unable to accumulate lipids andtherefore producing smaller lipid bodies.

qPCR measurements showed that all four promoters produce lowertranscript levels at 16 h than the native promoter (Table 3). Thisresult agreed with the lower fraction of Δ9 desaturated fatty acidsmeasured at this time point (FIG. 9B). In the wild-type strain, bothOLE1 transcript levels and Δ9 desaturated fatty acid levels wereslightly reduced at 96 h. OLE1 transcript levels for promoters PR105,PR109 and PR110 at 96 h were greatly reduced compared to 16 h values andcompared to YB-392. This result was also in agreement with GCcomposition data which showed lower Δ9 desaturated fatty acids for thesestrains at 96 h than 16 h and also a greater reduction of D9 desaturatedfatty acids between the time points than YB-392 (FIG. 9B and Table 3).The very low OLE1 transcript levels measured at 96 h for PR105, PR109and PR110 strains is also responsible for the small size of lipid bodiesobserved in these cells (FIG. 9C).

TABLE 3 OLE1 transcript levels of transformants and control strainmeasured by qPCR. All calculations were carried out using 2-ΔΔCt method.ratio: OLE1 transcript OLE1 transcript OLE1 ratio: relative to YB-relative to YB- transcript Δ9 products Strain 392 at 16 h 392 at 96 h at16 h/96 h at 16 h/96 h PR104  9.8%   11%  1 1.4 PR105  23% 0.44%  63 1.8PR109  20% 0.39%  63 1.8 PR110  38% 0.07% 688 1.8 YB-392 100%  100%  1.21.1 (wt)

C. Methods

1. Strains and Media

Wild-type Yarrowia lipolytica strain YB-392 was obtained from the ARSCulture Collection (NRRL). All strains were cultured in YPD (10 g/Lyeast extract, 20 g/L bacto peptone, and 20 g/L glucose) at 30° C. 20g/L agar was added to prepare solid media. Antibiotic selection wasachieved with the addition of hygromycin B (300 μg/mL) or nourseothricin(500 μg/mL) as appropriate. The lipid production media containing 0.5g/L urea, 1.5 g/L yeast extract, 0.85 g/L casamino acids, 1.7 g/L YNB(without amino acids and ammonium sulfate), 100 g/L glucose, and 5.11g/L potassium hydrogen phthalate (25 mM) was used for characterizingstrains in plates and flasks. 1.5 mL of media was used per well for24-well plates and 300 μl of media was used per well for 96-well plates.Alternatively, the yeast cultures were used to inoculate 50 ml ofsterilized media in an autoclaved 250 mL flask. Yeast strains that hadbeen incubated for 1-2 days on YPD-agar plates at 30° C. were used toinoculate each well of the multiwell plate.

2. Fatty Acid Composition Measurement

To obtain a lipid composition profile, a plate transesterificationprocedure was developed to extract and convert lipids to FAMEs. Samplesfrom shake flasks were pipetted into the wells of a 96-well plate andafter pelleting by centrifugation, cells were washed with water. Fluffypellets were made by vortexing the washed cells with a small amount ofwater and freezing at −80° C. for 30 min before placing the entire platein a lyophilizer overnight. To each well 500 μL 1.25 M HCl in Methanol(Sigma®) was added and the plate was sealed closed and incubated at 85°C. for 1.5 h with mixing by pipetting at 30-min intervals. 1 mLisooctane and 0.5 mL water were then added to each well and mixed byvortexing. A sample of the FAME-containing isooctane layer was analyzedby gas chromatography and composition was determined as percent of totalpeak area (sum of C16:0, C16:1, C18:0, C18:1, and C18:2) for each FAMEspecies. Because the dry cell weight in each of the 96 wells is notmeasured, this method yields relative compositional analysis bycomparing peak areas within each sample and not quantitative fatty acidlevels. For samples from shake flasks, 1 ml sample was pelleted, washedwith water and frozen at −80° C. for 30 min and extraction and GCanalysis was carried out in a similar manner as described above.

3. Targeted Deletions

Targeted genomic integrations to construct background strains NS418 andNS419 are previously described (Tsakraklides et al., Biotechnology forBiofuels, 2018). To delete Y. lipolytica genes OLE1 and FAD2 and toconstruct strains NS418 and NS419 respectively, the hygromycinselectable marker gene was amplified by PCR using oligonucleotideprimers that attach short flanks homologous to the promoter andterminator of target genes immediately 5′ and 3′ to the ORF incombination with internal marker gene primers. A two-fragment deletioncassette was thus made for each target such that the fragmentsoverlapped in the marker reading frame, but neither fragment alonecontained the entire functional antibiotic-resistance gene. PCR productswere transformed into hydroxyurea (Sigma-Aldrich®)-treated cells.

4. Gene Overexpression

Using standard molecular biology techniques, linear expressionconstructs were prepared. Each expression construct contained anexpression cassette for the gene of interest (OLE1 or FAD2) driven bythe promoters (PR104-PR110) described in this study, in tandem with anexpression cassette for the nourseothricin selectable marker. Theseconstructs were transformed into corresponding deletion mutant Y.lipolytica strains (Δole1 or Δfad2) leading to random integration intothe genome which can lead to expression differences. 10-12 transformantsselected on YPD/nourseothricin plates were grown in lipid productionmedia for 96 h in 96-deep well plates and the cell pellets were analyzedby Gas Chromatography. From this screen, two average transformants werechosen for a similar analysis in 24-well plates and finally one of thosetransformants was chosen to be tested in shake flasks. The cells fromshake flasks were tested for lipid composition and transcript levels byqPCR.

5. Transformation

For targeted integrations to delete endogenous genes FAD2 and OLE1,log-phase Y. lipolytica cells were treated with 50 mM hydroxyurea for 2h (targeted integration). For overexpression of FAD2 and OLE1 genesdriven by promoters PR104-110, Y. lipolytica cells were processeddirectly for transformation (random integration). Cells were washed withwater and resuspended in a volume of water equal to the wet cell pellet.50 μL was aliquoted per transformation reaction. 18 μL of desired DNAand 92 μL of transformation mix (80 μL 60% PEG4000, 5 μL 2 M DTT, 5 μL 2M lithium acetate pH 6, and 2 μL 10 mg/mL single stranded salmon spermDNA) were added to the cell pellet. The transformation reaction wasmixed by vortexing and heat shocked at 39° C. for 1 h. Cells werecentrifuged, the supernatant was discarded, and cells were resuspendedin 1 mL of a suitable non-selective medium like YPD, transferred toculture tubes, and cultured overnight at 30° C. before plating 100 μl(random integration) or all the cells (targeted integration) onselective media.

6. RNA Extraction and Transcript Quantification

Shake flask cultures grown in lipid production media were sampled at 16h and 96 h at an OD600 of 20 and the cell pellets were stored at −80° C.Total RNA was extracted using TRIzol™ reagent (Invitrogen®, cat. no.15596026, Pub. no. MAN0001271). Genomic DNA was removed using DNase(Qiagen®, Qiagen, cat no. 79254) followed by RNA cleanup using theQiagen RNeasy Mini Kit (Cat.no. 74134). 500 ng of clean RNA was used forcDNA synthesis using M-MuLV Reverse Transcriptase enzyme (NEB®, Cat No.M0253S). PCR quantification was done using PerfeCTa® SYBR® qPCR mix withHigh ROX as a reference dye by QuantaBio (Cat.No. 95055). The reactionswere run in PCR-96-LP-AB-C plates by Corning on the Applied Biosystems'StepOnePlus. Software analysis was performed with Applied Biosystems®StepOne v.2.3 software. YlACT1 encoding for actin was used as areference gene and gene expression levels were standardized using actingene as the reference (ΔCT method). For comparisons to YB-392 at 16 hand 96 h, the 2-ΔΔCT method was used to calculate fold difference andthen converted to percent change. The 2-ΔΔCT method was also used formeasuring fold repression for each strain, between the 16 h time pointand 96 h time point. The qPCR primers are listed in Table 4.

TABLE 4 List of qPCR primers Primer Sequence OLE1 qPCR_FCACAACCTGCTTGCCACCATG OLE1 qPCR_R CAGCTCGGTGGATTCGGTTTC FAD2 qPCR_FCCATGACATCATCGAGACCCACG FAD2 qPCR_R GTTGGTGTCGTCGTGTCGGTAG ACT1 qPCR_FGCTGCTGGTATCCACGAGACTAC ACT1 qPCR_R GCGGAGATCTCCTTGTGCATTCG

All of the methods disclosed and claimed herein can be made and executedwithout undue experimentation in light of the present disclosure. Whilethe compositions and methods of this disclosure have been described interms of preferred embodiments, it will be apparent to those of skill inthe art that variations may be applied to the methods and in the stepsor in the sequence of steps of the method described herein withoutdeparting from the concept, spirit and scope of the disclosure. Morespecifically, it will be apparent that certain agents which are bothchemically and physiologically related may be substituted for the agentsdescribed herein while the same or similar results would be achieved.All such similar substitutes and modifications apparent to those skilledin the art are deemed to be within the spirit, scope and concept of thedisclosure as defined by the appended claims.

The references recited in the application, to the extent that theyprovide exemplary procedural or other details supplementary to those setforth herein, are specifically incorporated herein by reference.

REFERENCES

The following references and the publications referred to throughout thespecification, to the extent that they provide exemplary procedural orother details supplementary to those set forth herein, are specificallyincorporated herein by reference.

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What is claimed is:
 1. A nucleic acid molecule comprising: a firstsequence having at least 80% sequence identity to SEQ ID NO:1, 2, 5, 6or 7, wherein the first sequence has promoter activity in a yeast cell;and a second sequence operably linked to the first sequence, wherein thesecond sequence is heterologous to the first sequence, and wherein thesecond sequence comprises a coding sequence.
 2. A yeast cell comprisingthe nucleic acid molecule of claim
 1. 3. The yeast cell of claim 2,wherein the yeast cell is an Arxula adeninivorans, Candida utilis,Hansenula polymorpha, Kluyveromyces lactis, Kluyveromyces marxianus,Kodamaea ohmeri, Lipomyces lipofer, Lipomyces starkeyi, Lipomycestetrasporus, Ogataea polymorpha, Pichia ciferrii, Pichia guilliermondii,Pichia pastoris, Pichia stipites, Rhodosporidium babjevae,Rhodosporidium toruloides, Rhodosporidium paludigenum, Rhodotorulaglutinis, Rhodotorula mucilaginosa, Saccharomyces cerevisiae,Schizosaccharomyces pombe, Wickerhamomyces ciferrii or Yarrowialipolytica cell.
 4. The yeast cell of claim 3, wherein the yeast cell isa Yarrowia lipolytica.
 5. A method comprising culturing the yeast cellof claim 2 under conditions sufficient to express the coding sequence.6. The method of claim 5, wherein the cell is cultured for at least 10passages.
 7. The method of claim 5, wherein the coding sequencecomprises SCT1.
 8. The method of claim 5, wherein yeast cell is aYarrowia lipolytica.
 9. The nucleic acid molecule of claim 1, whereinthe first sequence comprises SEQ ID NO:1, 2, 5, 6 or
 7. 10. The nucleicacid molecule of claim 1, wherein the second sequence is not fromYarrowia lipolytica.
 11. The nucleic acid molecule of claim 1, whereinthe second sequence comprises SCT1.
 12. The nucleic acid molecule ofclaim 1, wherein the first sequence is a subsequence of SEQ ID NO:1, 2,5, 6 or
 7. 13. The nucleic acid molecule of claim 1, wherein the firstsequence is capable of inducing increased expression of the secondsequence in a growth phase of a yeast cell relative to a lipidaccumulation phase of the yeast cell.
 14. The nucleic acid molecule ofclaim 1, wherein the first sequence has at least 85% sequence identityto SEQ ID NO:1, 2, 5, 6 or
 7. 15. The nucleic acid molecule of claim 1,wherein the first sequence has at least 90% sequence identity to SEQ IDNO:1, 2, 5, 6 or
 7. 16. The nucleic acid molecule of claim 1, whereinthe first sequence has at least 95% sequence identity to SEQ ID NO:1, 2,5, 6 or
 7. 17. The nucleic acid molecule of claim 1, wherein the yeastcell is a Yarrowia lipolytica.
 18. A method of optimizing lipidproduction of a cell during a fermentation process, wherein thefermentation process comprises a growth phase and a lipid accumulationphase, the method comprising: (a) introducing into a cell a nucleic acidmolecule comprising a promoter sequence having at least 80% sequenceidentity to SEQ IQ NO: 1, 2, 5, 6, or 7 to obtain a recombinant cellcomprising the promoter sequence operably linked to a coding sequence,wherein the coding sequence is heterologous to the promoter sequence;and (b) culturing the recombinant cell.
 19. The method of claim 18,wherein the coding sequence comprises SCT1.
 20. A method of regulatingthe expression or activity of a coding sequence during a fermentationprocess wherein the fermentation process comprises a growth phase and alipid accumulation phase, the method comprising: (a) introducing anucleic acid molecule into a yeast cell, wherein the nucleic acidmolecule comprises the coding sequence operably linked to a promotersequence, wherein the coding sequence is heterologous to the promotersequence, and wherein the promoter sequence has at least 80% sequenceidentity to SEQ ID NO: 1, 2, 5, 6, or 7; and (b) culturing the cell,thereby regulating expression or activity of the coding sequence. 21.The method of claim 20, wherein the expression or activity of the codingsequence during the lipid accumulation phase is reduced by at least 90%as compared to the growth phase.
 22. The method of claim 20, wherein thecell is selected from the group consisting of Arxula adeninivorans,Candida utilis, Hansenula polymorpha, Kluyveromyces lactis,Kluyveromyces marxianus, Kodamaea ohmeri, Lipomyces hpofer, Lipomycesstarkeyi, Lipomyces tetrasporus, Ogataea polymorpha, Pichia ciferrii,Pichia guilliermondii, Pichia pastoris, Pichia stipites, Rhodosporidiumbabjevae, Rhodosporidium toruloides, Rhodosporidium paludigenum,Rhodotorula glutinis, Rhodotorula mucilaginosa, Saccharomycescerevisiae, Schizosaccharomyces pombe, Wickerhamomyces ciferrii, andYarrowia lipolytica.